Antibodies against human cd38

ABSTRACT

Isolated monoclonal antibodies which bind to human CD38 and related antibody-based compositions and molecules, are disclosed. Also disclosed are pharmaceutical compositions comprising the antibodies and therapeutic and diagnostic methods for using the antibodies.

FIELD OF THE INVENTION

The present invention relates to antibodies directed to human CD38 andto uses of such antibodies, in particular therapeutic uses.

BACKGROUND OF THE INVENTION

CD38 is a type II transmembrane glycoprotein which is normally found onhemopoietic cells and in solid tissues. With regard to hemopoieticcells, the majority of medullary thymocytes are CD38⁺, resting andcirculating T- and B-cells are CD38⁻ and activated cells are CD38⁺. CD38is also expressed on approximately 80% of resting NK cells and monocytesand on lymph node germinal center lymphoblasts, plasma B cells and someintrafollicular cells. CD38 can also be expressed by dendritic cells. Asignificant proportion of normal bone marrow cells, particular precursorcells, express CD38. In addition, 50-80% of umbilical cord blood cellsis CD38⁺ and remains so in human blood for the first two to three yearsof life. In addition to lymphoid precursor cells, CD38 is also expressedon erythrocytes and on platelets. With regard to solid tissues, CD38 isexpressed in the gut by intra-epithelial cells and lamina proprialymphocytes, by Purkinje cells and neurofibrillary tangles in the brain,by epithelial cells in the prostate, β-cells in the pancreas,osteoclasts in the bone, retinal cells in the eye, and sarcolemma ofsmooth and striated muscle.

CD38 is also expressed in a variety of malignant hematological diseases,including multiple myeloma, B-cell chronic lymphocytic leukemia, B-cellacute lymphocytic leukemia, Waldenström macroglobulinemia, primarysystemic amyloidosis, mantle-cell lymphoma, pro-lymphocytic/myelocyticleukemia, acute myeloid leukemia, chronic myeloid leukemia, follicularlymphoma, NK-cell leukemia and plasma-cell leukemia. Expression of CD38has been described on epithelial/endothelial cells of different origin,including glandular epithelium in prostate, islet cells in pancreas,ductal epithelium in glands, including parotid gland, bronchialepithelial cells, cells in testis and ovary and tumor epithelium incolorectal adenocarcinoma. Other diseases, where CD38 expression couldbe involved, include, e.g. broncho-epithelial carcinomas of the lung,breast cancer (evolving from malignant proliferation of epitheliallining in ducts and lobules of the breast), pancreatic tumors, evolvingfrom the b-cells (insulinomas), tumors evolving from epithelium in thegut (e.g. adenocarcinoma and squamous cell carcinoma), carcinoma in theprostate gland, seminomas in testis and ovarian cancers. In CNS,neuroblastomas express CD38.

Other disclosures also suggest the role of CD38 in autoimmunity such asGraves disease and thyroiditis (Antonelli A, et. al., Clin. Exp.Immunol. 126, 426-431, 2001), and type 1 and 2 Diabetes (Mallone R andPerin P C, Diabetes Metab Res Rev 2006; 22: 284-294) and inflammation ofairway smooth muscle cells during asthma (Desphande et al. 2004 am JRespir Cell Mol Biol 31: 36-42)

CD38 is a multifunctional protein. Functions ascribed to CD38 includeboth receptor mediation in adhesion and signaling events and (ecto-)enzymatic activity. As an ectoenzyme, CD38 uses NAD⁺ as substrate forthe formation of cyclic ADP-ribose (cADPR) and ADPR, but also ofnicotinamide and nicotinic acid-adenine dinucleotide phosphate (NAADP).cADPR has been shown to act as second messenger for Ca²⁺ mobilizationfrom the endoplasmatic reticulum. The CD38/cyclic ADP ribose system: 1)in lung, contributes to airway smooth muscle tone and responsivenessthrough its effects on agonist induced elevation of intra-cellular Ca²⁺(Desphande et al. 2005 Am J physiol Lung cell Mol Physiol 288:L773-L788), 2) regulates migration of neutrophil chemotaxis to bacterialchemoattractants, migration of DC precursors from blood to peripheralsites and migration of mature DCs from sites of inflammation to lymphnodes (Partida-Sanchez et al. Nat Med 7: 1209-121, 2001; Morita et al.2008 J Pharmacol Sci. 2008 March; 106(3):492-504; Partida-Sanchez et al.Immunity 20: 279-291, 2004), 3) is involved in astrocyte calciumsignaling which has implications for neuroinflammation andHIV-1-associated dementia (Banerjee S. et. al., J. NeurimmunePharmacol., 3, 154-164 (2008)), 4) regulates FcγR-mediated phagocytosisin murine macrophages (Song E., et. al., Biochem. and Biophys. Res.Comm., 367, 156-161, (2008), 5) is linked to insulin secretion Okamoto,Molecular and Cellular Biochemistry, 193, 115-118, 1999 and 6) has a keyrole in neuropeptide release and regulating maternal and socialbehaviors (Jin D et al. Nature 446: 41-45, 2007). In addition tosignaling via Ca²⁺, CD38 signaling occurs via cross-talk withantigen-receptor complexes on T and B cells or other types of receptorcomplexes, e.g. MHC molecules, and is in this way involved in severalcellular responses, but also in switching and secretion of IgG1.

Several anti-CD38 antibodies are described in the literature, forinstance in Lande R, et al., Cell Immunol. 220(1), 30-8 (2002), AusielloC M, et al., Tissue Antigens. 56(6), 539-47 (2000), and Cotner T, etal., Int J Immunopharmacol. 3(3), 255-68 (1981). Antibody binding toCD38 can have different effects on the functions of CD38. For instance,mouse anti-CD38 antibody IB4 has been shown to induce T cell activationas indicated by Ca²⁺ mobilization in Jurkat cells (Zubiaur M, et al., JImmunol. 159(1), 193-205 (1997), to induce significant proliferation ofperipheral blood mononuclear cells (PBMCs), to induce release ofsignificant IL-6 levels and to induce release of detectable IFN-γ levels(Lande, Zubiaur Morra, Ansiello supra). Hara-Yokoyama et al. IntImmunopharmacol 8, 59-70 (2008) described one anti-mouse CD38 antibody(CS/2) which inhibits the NAD⁺ glycohydrolase activity of CD38 andanother anti-mouse CD38 antibody (clone 90) which stimulates the NAD⁺glycohydrolase activity of an isolated extracellular domain of CD38, buthas little effect on the NAD⁺ glycohydrolase activity of cell-surfaceCD38. As it can be seen from data presented below, the antibodies of thepresent invention provide activity on the surface of CD38 positivecells.

WO2006099875 (Genmab) describes several human anti-CD38 antibodies,including 003 and 005. Antibody 005 was shown to inhibit the productionof cGDPR from NGD⁺ by CD38.

In view of the multiple functions of human CD38, there is a need for newtherapeutic antibodies that more specifically modulate particularfunctions of CD38.

SUMMARY OF THE INVENTION

The present invention provides a new class of anti-CD38 antibodies whichthrough interacting with particular amino acids of human CD38 have astrong stimulating effect on the cADPR hydrolase activity of CD38leading to decreased levels of cADPR. Furthermore, the anti-CD38antibodies inhibit the ability of CD38 to catalyze the formation, via abase-exchange reaction, of nicotinic acid adenine dinucleotide2′-phosphate (NAADP).

These antibodies are useful for the treatment of several diseases,including autoimmune and (chronic) inflammatory diseases, such as Type 1and 2 diabetes, thyroiditis, Graves disease, arthritis,neuroinflammation and asthma.

Recent scientific work suggests that cADPR synthesized extracellularlyby CD38, may be transported into cells through nucleoside transportersand then mobilize Ca(2+) through a FK506-binding protein-dependentprocess. This process may be involved in fMLP-induced intracellularCa(2+) signaling and migration in human neutrophils (Morita et al. 20083 Pharmacol Sci. 2008 March; 106(3):492-504), migration of DC precursorsfrom blood to peripheral sites and migration of mature DCs from sites ofinflammation to lymph nodes (Partida-Sanchez et al. Immunity 20:279-291, 2004). Without being bound by any particular theory, thereduction of cADPR levels obtained by treatment with an antibody of thepresent invention may thus reduce migration of neutrophils and dendriticcells and have anti-inflammatory effects. Accordingly, while theantibodies of the invention may be useful for a number of purposes, theymay be particularly useful for the treatment of inflammation, e.g. inconnection with autoimmune disease, because of their unique effects onthe enzymatic activities of CD38, through binding at a particular siteon CD38.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cross-block studies of antibodies of the invention. Moreparticularly, the figure shows the binding of 005-FITC to CHO-CD38 cellstreated with excess unlabelled CD38-specific antibodies 025, 026, 028,049 and 056.

FIG. 2 shows binding of the anti-CD38 antibodies of the invention to wtand mutant CD38. FIG. 2(A) shows binding of the anti-CD38 antibodies025, 026, 028 and 049 to wild type (WT) and mutant (T237A, Q272R, andS274F) CD38. FIG. 2(B) shows binding of the anti-CD38 antibodies 025,028 and 049 to wild type (WT) and mutant (D202G) CD38.

FIG. 3 shows binding of antibodies of the invention to Daudi-luc cellsand CHO-CD38 cells.

FIG. 4 shows ADCC mediated lysis of Daudi-luc cells caused by theanti-CD38 antibodies of the invention and as isotype control anti-KLHantibody (HuMab-KLH.

FIG. 5 shows CDC mediated lysis of CHO CD38 cells caused by theanti-CD38 antibodies of the invention.

FIG. 6 shows inhibition of cGDPR production by His-tagged CD38 proteinand cellular expressed CD38 in the presence of the anti-CD38 antibodiesof the invention. FIG. 6(A) shows the percentage inhibition of cGDPRproduction (by recombinant human CD38 protein) in the presence of CD38specific antibodies 025, 026, 028, 049 and 056 (3 μg/mL). FIG. 6(B)shows the effect of the anti-CD38 antibodies on cGDPR production intime. The anti-CD38 antibodies were used at a final concentration of 10μg/ml. FIG. 6(C) shows the effect of the anti-CD38 antibodies on cGDPRproduction using serial dilutions (0.01-30 μg/mL) of 028 or isotypecontrol HuMab-KLH. FIG. 6(D) shows the percentage inhibition of cGDPRproduction (by cellular expressed CD38 (CHO-CD38 cells)) in the presenceof serial dilutions (0.01-30 μg/mL) of 028 or IgG1 isotype controlHuMab-KLH.

FIG. 7 shows the effect of antibody 028 of the invention on 8NH2-cADPRproduction. Products of each reaction were analyzed by HPLC. FIG. 7(A)indicates the elution position of the products and substrates. FIG. 7(B)shows the antibody concentration dependence on 8NH2-cADPR production.HuMab-KLH (open circles), mAb-028 (closed circles).

FIG. 8 shows the effect of antibody 028 of the invention on cADPRhydrolase and NADa se activity, more particularly the effect of mAb-028on cADPR hydrolase (Figure A, left figure, B and C) and NADse (Figure A,right figure) activity. FIG. 8(A) shows the results of incubating CD38recombinant protein with cADPR or NAD in the presence of 10 μg HuMab-KLH(CD38+10 μg HuMab-KLH), 10 μg Ab 028 (CD38+10 μg Ab 028), or no antibody(CD38 control). Products of each reaction were analyzed by HPLC. FIG.8(B) shows CD38 antibody titration at different concentrations on cADPRhydrolase activity as analyzed by HPLC. FIG. 8(C) shows the results ofincubating CD38 recombinant protein with ³²P-cADPR in the presence ofmAb-003, mAb-028, daratumumab (005), or HuMab-KLH. Products wereanalyzed by thin layer chromatography. HuMab-KLH (open circles), mAb-028(closed circles).

FIG. 9 shows the effect of the anti-CD38 antibodies of the invention onthe base-exchange activity of CD38. FIG. 9(A) shows the effect of theantibodies on NAADP production at the indicated concentrations. FIG.9(B) shows the effect of mAb-028 titration on the rate of NAADPformation.

SEQUENCE LIST VH-region SEQ ID VH 028 DNA NO: 1caggtccaac tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtctcctgcaagg cttttggagg caccttcagc agctacgcta tcagctgggt gcgacaggcccctggacaag ggcttgagtg gatgggaagg atcatccgtt tccttggtat agcaaactacgcacagaagt tccagggcag agtcacgctt atcgcggaca aatccacgaa cacagcctacatggagctga gcagcctgag atctgaggac acggccgttt attactgtgc gggggaacctggggagcggg accccgatgc tgttgatatc tggggccaag ggacaatggt caccgtctct tcaSEQ ID VH 028 NO: 2 QVQLVQSGAE VKKPGSSVKV SCKAFGGTFSSYAISWVRQA PGQGLEWMGR IIRFLGIANY AQKFQGRVTL IADKSTNTAY MELSSLRSEDTAVYYCAGEP GERDPDAVDI WGQGTMVTVSS SEQ ID VH 028 CDR1 NO: 3 GGTSFSSYASEQ ID VH 028 CDR2 NO: 4 IIRFLGIA SEQ ID VH 028 CDR3 NO: 5AGEPGERDPDAVDI SEQ ID VH 025 DNA NO: 6caggtccaactggtgcagtctggggctgaggtga agaagcctgggtcctcggtgaaggtctcctgcaaggcttttggaggcaccttcagcagctatgcta tcagctgggtacgacaggcccctggacaagggcttgagtggatgggaaggatcatccgtttccttg gtaaagcaaatcacgcacagaagttccagggcagagtcacgcttaccgcggacaaatccacgaaca cagcctacatggagctgagcagcctgagatctgaggacacggccgtttattactgtgcgggggaac ctggggatcgggaccccgatgctgttgatatctggggccaagggacaatggtcaccgtctcttcag SEQ ID VH 025 NO: 7QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYA ISWVRQAPGQGLEWMGRIIRFLGKANHAQKFQGRVTLTADKSTNTAYMELSSLRSEDTAVYYCAGE PGDRDPDAVDIWGQGTMVTVSS SEQ IDVH 025 CDR1 NO: 8 GGTFSSYA SEQ ID VH 025 CDR2 NO: 9 IIRFLGKA SEQ IDVH 025 CDR3 NO: 10 AGEPGDRDPDAVDI SEQ ID VH 026 DNA NO: 11caggtccaactggtgcagtctggggctgaggt gaagaagcctgggtcctcggtgaaggtctcctgcaaggcttttggaggcaccttcagcagttat gctattagctgggtgcgacaggcccctggacaagggcttgagtggatgggaaggatcatccgtt tccttggtaaaacaaatcacgcacagaagttccagggcagagtcacacttaccgcggacaaatc cacgaacacagcctacatggagctgagcagcctgagatctgaggacacggccgtttattactgt gcgggggaacctggggatcgggaccccgatgctgttgatatctggggccaagggacaatggtca ccgtctcttcag SEQ ID VH 026 NO: 12QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYA ISWVRQAPGQGLEWMGRIIRFLGKTNHAQKFQGRVTLTADKSTNTAYMELSSLRSEDTAVYYCAGE PGDRDPDAVDIWGQGTMVTVSS SEQ IDVH 026 CDR1 NO: 13 GGTFSSYA SEQ ID VH 026 CDR2 NO: 14 IIRFLGKT SEQ IDVH 026 CDR3 NO: 15 AGEPGDRDPDAVDI SEQ ID VH 049 DNA NO: 16caggtccagctggtgcagtctggggctgaggtg atgaagcctgggtcctcggtgaaggtctcctgcaaggcttccggaggcaccttccgcagctatgct atcagttgggtgcgacaggcccctggacaagggcttgagtggatgggaaggatcatcgttttcctt ggtaaaacaaactacgcacagaagttccagggcagagtcacgcttaccgcggacaaatccacgacc acagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtacgggggaa cctggggctcgggaccccgacgcttttgatatctggggccaagggacaatggtcaccgtctcttca g SEQ ID VH 049 NO: 17QVQLVQSGAEVMKPGSSVKVSCKASGGTFRSYA ISWVRQAPGQGLEWMGRIIVFLGKTNYAQKFQGRVTLTADKSTTTAYMELSSLRSEDTAVYYCTGE PGARDPDAFDIWGQGTMVTVSS SEQ IDVH 049 CDR1 NO: 18 GGTFRSYA SEQ ID VH 049 CDR2 NO: 19 IIVFLGKT SEQ IDVH 049 CDR3 NO: 20 TGEPGARDPDAFDI SEQ ID VH 056 DNA NO: 21caggtccagctggtgcagtctggggctgaggtg aagaagcctgggtcctcggtgaaggtctcctgcaagccttccggaggcaccttcaggagctacgct atcagctgggtacgacaggcccctggacaagggcttgagtggatgggaaggatcatcgttttcctt ggtaaagtaaactacgcacagaggtttcagggcagagtcacgcttaccgcggacaaatccacgacc acagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtacgggggaa cctggggctcgggaccccgacgcttttgatatctggggccaagggacaatggtcaccgtctcttca g SEQ ID VH 056 NO: 22QVQLVQSGAEVKKPGSSVKVSCKPSGGTFRSYA ISWVRQAPGQGLEWMGRIIVFLGKVNYAQRFQGRVTLTADKSTTTAYMELSSLRSEDTAVYYCTGE PGARDPDAFDIWGQGTMVTVSS SEQ IDVH 056 CDR1 NO: 23 GGTFRSYA SEQ ID VH 056 CDR2 NO: 24 IIVFLGKV SEQ IDVH 056 CDR3 NO: 25 TGEPGARDPDAFDI SEQ ID VL 028 DNA NO: 26gacatccagatgacccagtctccatcctcactg tctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattcgcagctggtta gcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaa agtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgccaacagtataatagttacccgctcactttcggc ggagggaccaaggtggagatcaaa SEQ ID VL 028NO: 27 DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK SEQ ID VL 028 CDR1 NO: 28 GGIRSW SEQ ID VL 028 CDR2 NO: 29 AASSEQ ID VL 028 CDR3 NO: 30 QQYNSYPLT SEQ ID VL 025 DNA NO: 31gacatccagatgacccagtctccatcctcactg tctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattcgcagctggtta gcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaa agtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgccaacagtataatagttacccgctcactttcggc ggagggaccaaggtggagatcaaac SEQ IDVL 025 NO: 32 DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK SEQ ID VL 025 CDR1 NO: 33 QGIRSW SEQ ID VL 025 CDR2 NO: 34 AASSEQ ID VL 025 CDR3 NO: 35 QQYNSYPLT SEQ ID VL 026 DNA NO: 36gacatccagatgacccagtctccatcctcactg tctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattcgcagctggtta gcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaa agtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgccaacagtataatagttacccgctcactttcggc ggagggaccaaggtggagatcaaac SEQ IDVL 026 NO: 37 DIQMTQSPSSLSASVGDRVnTCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK SEQ ID VL 026 CDR1 NO: 38 QGIRSW SEQ ID VL 026 CDR2 NO: 39 AASSEQ ID VL 026 CDR3 NO: 40 QQYNSYPLT SEQ ID VL 049 DNA NO: 41gacatccagatgacccagtctccatcctcactg tctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattcgcagctggtta gcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaa agtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgccaacagtataataattatccgctcactttcggc ggagggaccaaggtggagatcaaac SEQ IDVL 049 NO: 42 DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIK SEQ ID VL 049 CDR1 NO: 43 QGIRSW SEQ ID VL 049 CDR2 NO: 44 AASSEQ ID VL 049 CDR3 NO: 45 QQYNNYPLT SEQ ID VL 056 DNA NO: 46gacatccagatgacccagtctccatcctcactg tctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattcgcagctggtta gcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaa agtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagc ctgcagcctgaagattttgcaacttattactgccaacagtataataattatccgctcactttcggc ggagggaccaaggtggagatcaaac SEQ IDVL 056 NO: 47 DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIK SEQ ID VL 056 CDR1 NO: 48 QGIRSW SEQ ID VL 056 CDR2 NO: 49 AASSEQ ID VL056CDR3 NO: 50 QQYNNYPLT SEQ ID Mutant human CD38 NO: 51MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVL ILVVVLAVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPC NITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNT SKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQ PEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCT SEI SEQ ID Human CD38 NO: 52MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVL ILVVVLAVVVPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPC NITEEDYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNT SKINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQ PEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCT SEI

CDR regions are indicated according to IMGT.

The sequence of human CD38 is described in sequence 52. A mutant ofhuman CD38 wherein S was mutated to F at position 274 was described inWO2006099875 as SEQ. ID NO: 34, and a mutation wherein Q was mutated toR at position 272 was described in WO2006099875 as SEQ. ID NO: 33. Amutant of human CD38 wherein D was mutated to G at position 202 isdescribed above as SEQ. ID NO: 51.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “human CD38” when used herein includes any variants, isoformsand species homologs of human CD38 (Swissprot: locus CD38 HUMAN,accession P28907) which are naturally expressed by cells or areexpressed on cells transfected with the human CD38 gene.

The term “immunoglobulin” refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,all four inter-connected by disulfide bonds. The structure ofimmunoglobulins has been well characterized. See for instanceFundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). Briefly, each heavy chain typically is comprised of a heavychain variable region (abbreviated herein as V_(H) or VH) and a heavychain constant region. The heavy chain constant region typically iscomprised of three domains, C_(H)1, C_(H)2, and C_(H)3. Each light chaintypically is comprised of a light chain variable region (abbreviatedherein as V_(L) or VL) and a light chain constant region. The lightchain constant region typically is comprised of one domain, C_(L). TheV_(H) and V_(L) regions may be further subdivided into regions ofhypervariability (or hypervariable regions which may be hypervariable insequence and/or form of structurally defined loops), also termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FRs). Each V_(H) andV_(L) is typically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196,901-917 (1987)). Typically, the numbering of amino acid residues in thisregion is performed by the method described in Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) (phrases such asvariable domain residue numbering as in Kabat or according to Kabatherein refer to this numbering system for heavy chain variable domainsor light chain variable domains). Using this numbering system, theactual linear amino acid sequence of a peptide may contain fewer oradditional amino acids corresponding to a shortening of, or insertioninto, a FR or CDR of the variable domain. For example, a heavy chainvariable domain may include a single amino acid insert (residue 52aaccording to Kabat) after residue 52 of V_(H) CDR2 and inserted residues(for instance residues 82a, 82b, and 82c, etc. according to Kabat) afterheavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule, a fragment of an immunoglobulin molecule,or a derivative of either thereof, which has the ability to specificallybind to an antigen under typical physiological conditions with a halflife of significant periods of time, such as at least about 30 minutes,at least about 45 minutes, at least about one hour, at least about twohours, at least about four hours, at least about 8 hours, at least about12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5,6, 7 or more days, etc., or any other relevant functionally-definedperiod (such as a time sufficient to induce, promote, enhance, and/ormodulate a physiological response associated with antibody binding tothe antigen and/or time sufficient for the antibody to recruit anFc-mediated effector activity). The variable regions of the heavy andlight chains of the immunoglobulin molecule contain a binding domainthat interacts with an antigen. The constant regions of the antibodies(Abs) may mediate the binding of the immunoglobulin to host tissues orfactors, including various cells of the immune system (such as effectorcells) and components of the complement system such as C1q, the firstcomponent in the classical pathway of complement activation. Ananti-CD38 antibody may also be a bispecific antibody, diabody, orsimilar molecule (see for instance PNAS USA 90(14), 6444-8 (1993) for adescription of diabodies). Indeed, bispecific antibodies, diabodies, andthe like, provided by the present invention may bind any suitable targetin addition to a portion of CD38. As indicated above, the term antibodyherein, unless otherwise stated or clearly contradicted by context,includes fragments of an antibody that retain the ability tospecifically bind to the antigen. It has been shown that theantigen-binding function of an antibody may be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antibody” include (i) a Fab′ or Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), C_(L) and C_(H)1 domains, or amonovalent antibody as described in WO2007059782 (Genmab); (ii) F(ab′)₂fragments, bivalent fragments comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consistingessentially of the V_(H) and C_(H)1 domains; (iv) a Fv fragmentconsisting essentially of the V_(L) and V_(H) domains of a single arm ofan antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546(1989)), which consists essentially of a VH domain and also calleddomain antibodies (Holt et al; Trends Biotechnol. 2003 November;21(11):484-90); (vi) camelid or nanobodies (Revets et al; Expert OpinBiol Ther. 2005 January; 5(1):111-24) and (vii) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, V_(L) and V_(H), are coded for by separategenes, they may be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe V_(L) and V_(H) regions pair to form monovalent molecules (known assingle chain antibodies or single chain Fv (scFv), see for instance Birdet al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85,5879-5883 (1988)). Such single chain antibodies are encompassed withinthe term antibody unless otherwise noted or clearly indicated bycontext. Although such fragments are generally included within themeaning of antibody, they collectively and each independently are uniquefeatures of the present invention, exhibiting different biologicalproperties and utility. These and other useful antibody fragments in thecontext of the present invention are discussed further herein. It alsoshould be understood that the term antibody, unless specified otherwise,also includes polyclonal antibodies, monoclonal antibodies (mAbs),antibody-like polypeptides, such as chimeric antibodies and humanizedantibodies, and antibody fragments retaining the ability to specificallybind to the antigen (antigen-binding fragments) provided by any knowntechnique, such as enzymatic cleavage, peptide synthesis, andrecombinant techniques. An antibody as generated can possess anyisotype. As used herein, “isotype” refers to the immunoglobulin class(for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that isencoded by heavy chain constant region genes. An “anti-CD38 antibody” isan antibody which binds to the antigen CD38.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

In a preferred embodiment, the antibody of the invention is isolated. An“isolated antibody,” as used herein, is intended to refer to an antibodywhich is substantially free of other antibodies having differentantigenic specificities (for instance an isolated antibody thatspecifically binds to CD38 is substantially free of antibodies thatspecifically bind antigens other than CD38). An isolated antibody thatspecifically binds to an epitope, isoform or variant of human CD38 may,however, have cross-reactivity to other related antigens, for instancefrom other species (such as CD38 species homologs). Moreover, anisolated antibody may be substantially free of other cellular materialand/or chemicals. In one embodiment of the present invention, acombination of “isolated” monoclonal antibodies having differentspecificities is combined in a well-defined composition.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from human germline immunoglobulin sequences. The humanmonoclonal antibodies may be generated by a hybridoma which includes a Bcell obtained from a transgenic or transchromosomal nonhuman animal,such as a transgenic mouse, having a genome comprising a human heavychain transgene and a light chain transgene, fused to an immortalizedcell.

As used herein, the term “binding” in the context of the binding of anantibody to a predetermined antigen typically is a binding with anaffinity corresponding to a K_(D) of about 10⁻⁷ M or less, such as about10⁻⁸ M or less, such as about 10⁻⁹ M or less, about 10⁻¹⁰ M or less, orabout 10⁻¹¹ M or even less when determined by for instance surfaceplasmon resonance (SPR) technology in a BIAcore 3000 instrument usingthe antigen as the ligand and the antibody as the analyte, and binds tothe predetermined antigen with an affinity corresponding to a K_(D) thatis at least ten-fold lower, such as at least 100 fold lower, forinstance at least 1,000 fold lower, such as at least 10,000 fold lower,for instance at least 100,000 fold lower than its affinity for bindingto a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The amount withwhich the affinity is lower is dependent on the K_(D) of the antibody,so that when the K_(D) of the antibody is very low (that is, theantibody is highly specific), then the amount with which the affinityfor the antigen is lower than the affinity for a non-specific antigenmay be at least 10,000 fold.

The term “k_(d)” (sec⁻¹), as used herein, refers to the dissociationrate constant of a particular antibody-antigen interaction. Said valueis also referred to as the k_(off) value.

The term “k_(a)” (M⁻¹×sec⁻¹), as used herein, refers to the associationrate constant of a particular antibody-antigen interaction.

The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction.

The term “K_(A)” (M⁻¹), as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the k_(a) by the k_(d).

The antibodies of the present invention have an effect on enzymaticsystems as described in the examples section. The antibodies aredescribed by stimulatory effects or inhibitory effects on differentparameters. The stimulatory and inhibitory effects may be measured asdisclosed in the examples herein.

An antibody as described and claimed herein may also be a functionalvariant of any of the specific antibodies described herein. Such avariant antibody is an antibody that differs from a specific antibodydescribed herein by one or more suitable amino acid residue alterations,that is substitutions, deletions, insertions, or terminal sequenceadditions, for instance in the constant domain, and/or the variableregions (or any one or more CDRs thereof) in a single variant antibody.A functional variant of a V_(L), V_(H), or CDR region used in thecontext of an anti-CD38 antibody still allows the antibody to retain atleast a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%,95% or more) of the affinity/avidity and/or the specificity/selectivityof the parent antibody and in some cases such an anti-CD38 antibody maybe associated with greater affinity, selectivity and/or specificity thanthe parent antibody.

Such functional variants typically retain significant sequence identityto the parent antibody. The percent identity between two sequences is afunction of the number of identical positions shared by the sequences(i.e., % homology=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences may be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

The percent identity between two nucleotide sequences may be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences may alsobe determined using the algorithm of E. Meyers and W. Miller, Comput.Appl. Biosci 4, 11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences may be determined using the Needlemanand Wunsch, J. Mol. Biol. 48, 444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The sequence of CDR variants may differ from the sequence of the CDR ofthe parent antibody sequences through mostly conservative substitutions;for instance at least about 35%, about 50% or more, about 60% or more,about 70% or more, about 75% or more, about 80% or more, about 85% ormore, about 90% or more, about 95% or more (e.g., about 65-99%) of thesubstitutions in the variant are conservative amino acid residuereplacements. In the context of the present invention, conservativesubstitutions may be defined by substitutions within the classes ofamino acids reflected in one or more of the following three tables:

Amino Acid Residue Classes for Conservative Substitutions

Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg (R), andHis (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), andGln (Q) Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu (L),and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P)Aromatic Residues Phe (F), Tyr (Y), and Trp (W)

Alternative Conservative Amino Acid Residue Substitution Classes

1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

Alternative Physical and Functional Classifications of Amino AcidResidues

Alcohol group-containing residues S and T Aliphatic residues I, L, V,and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobicresidues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively chargedresidues D and E Polar residues C, D, E, H, K, N, Q, R, S, and TPositively charged residues H, K, and R Small residues A, C, D, G, N, P,S, T, and V Very small residues A, G, and S Residues involved in turn A,C, D, E, G, H, K, N, Q, R, S, P, and formation T Flexible residues Q, T,K, S, G, P, D, E, and R

More conservative substitutions groupings include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Additional groups of amino acids may also be formulated using theprinciples described in, e.g., Creighton (1984) Proteins: Structure andMolecular Properties (2d Ed. 1993), W.H. Freeman and Company.

As explained above, typically, amino acid sequence alterations,desirably do not substantially change the structural characteristics ofthe parent sequence (e.g., a replacement amino acid should not tend todisrupt secondary structure that characterizes the function of theparent sequence), but may be associated with advantageous properties,such as changing the functional or pharmacokinetic properties of theantibodies, for example increasing the half-life, altering theimmunogenicity, providing a site for covalent or non-covalent binding toanother molecule, reducing susceptibility to proteolysis, reducingsusceptibility to oxidation, or altering the glycosylation pattern.

Examples of functional properties of antibodies, which may be altered orretained in variant anti-CD38 antibodies of the present inventioncompared to antibodies of prior art are for example:

(1) high affinity binding to CD38 and/or(2) binding to transfected cells, e.g. CHO or HEK293 cells expressingCD38 and/or(3) induction of CDC and/or(4) induction of ADCC and/or(5) alteration of enzymatic activity and/or(6) induction of apoptosis after secondary cross-linking and/or(7) phagocytosis

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and nonconformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents. The epitope maycomprise amino acid residues directly involved in the binding (alsocalled immunodominant component of the epitope) and other amino acidresidues, which are not directly involved in the binding, such as aminoacid residues which are effectively blocked by the specifically antigenbinding peptide (in other words, the amino acid residue is within thefootprint of the specifically antigen binding peptide).

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, for instance by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library, and wherein the selected human antibody isat least 90%, such as at least 95%, for instance at least 96%, such asat least 97%, for instance at least 98%, or such as at least 99%identical in amino acid sequence to the amino acid sequence encoded bythe germline immunoglobulin gene. Typically, outside the heavy chainCDR3, a human antibody derived from a particular human germline sequencewill display no more than 20 amino acid differences, e.g. no more than10 amino acid differences, such as no more than 5, for instance no morethan 4, 3, 2, or 1 amino acid difference from the amino acid sequenceencoded by the germline immunoglobulin gene.

As used herein, the term “inhibits growth” (e.g. referring to cells,such as tumor cells) is intended to include any measurable decrease inthe cell growth when contacted with an anti-CD38 antibody as compared tothe growth of the same cells not in contact with an anti-CD38 antibody,e.g., the inhibition of growth of a cell culture by at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Such a decrease incell growth can occur by a variety of mechanisms, e.g. effector cellphagocytosis, ADCC, CDC, and/or apoptosis.

The term “bispecific antibody” is intended to include any antibody whichhas two different binding specificities. The term “bispecificantibodies” also includes diabodies (see for instance Holliger, P. etal., PNAS USA 90, 6444-6448 (1993), Poljak, R. J. et al., Structure 2,1121-1123 (1994)).

An “antibody deficient in effector function” or an“effector-function-deficient antibody” refers to an antibody which has asignificantly reduced or no ability to activate one or more immuneeffector mechanisms, such as complement activation or Fc receptorbinding. Thus, effector-function deficient antibodies have significantlyreduced or no ability to mediate antibody-dependent cell-mediatedcytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC).

The term “monovalent antibody” means in the context of the presentinvention that an antibody molecule is capable of binding a singlemolecule of the antigen, and thus is not able of antigen crosslinking.

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Exemplaryimmune cells include a cell of a myeloid or lymphoid origin, forinstance lymphocytes (such as B cells and T cells including cytolytic Tcells (CTLs)), killer cells, natural killer cells, macrophages,monocytes, eosinophils, polymorphonuclear cells, such as neutrophils,granulocytes, mast cells, and basophils. Some effector cells expressspecific Fc receptors and carry out specific immune functions. In someembodiments, an effector cell is capable of inducing antibody-dependentcellular cytotoxicity (ADCC), such as a natural killer cell, capable ofinducing ADCC. For example, monocytes, macrophages, which express FcRare involved in specific killing of target cells and presenting antigensto other components of the immune system, or binding to cells thatpresent antigens. In some embodiments, an effector cell may phagocytosea target antigen or target cell.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. Various types of vectors are well-known in the art. Onetype of vector is a plasmid.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which an expression vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell, but also tothe progeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, transfectomas,such as CHO cells, HEK293 cells, NS/0 cells, and lymphocytic cells.

The term “transgenic non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or transchromosomes (either integrated or non-integrated intothe animal's natural genomic DNA) and which is capable of expressingfully human antibodies. For example, a transgenic mouse can have a humanlight chain transgene and either a human heavy chain transgene or humanheavy chain transchromosome, such that the mouse produces humananti-CD38 antibodies when immunized with CD38 antigen and/or cellsexpressing CD38. The human heavy chain transgene may be integrated intothe chromosomal DNA of the mouse, as is the case for transgenic mice,for instance HuMAb mice, such as HCo7 or HCo12 mice, or the human heavychain transgene may be maintained extrachromosomally, as is the case fortranschromosomal KM mice as described in WO02/43478. Such transgenic andtranschromosomal mice (collectively referred to herein as “transgenicmice”) are capable of producing multiple isotypes of human monoclonalantibodies to a given antigen (such as IgG, IgA, IgM, IgD and/or IgE) byundergoing V-D-J recombination and isotype switching. Transgenic,nonhuman animal can also be used for production of antibodies against aspecific antigen by introducing genes encoding such specific antibody,for example by operatively linking the genes to a gene which isexpressed in the milk of the animal.

The terms “B-cell neoplasms” or “mature B-cell neoplasms” in the contextof the present invention include small lymphocytic lymphoma, B-cellprolymphocytic lymphoma, B-cell chronic lymphocytic leukemia, mantlecell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse largeB-cell lymphoma, multiple myeloma, lymphoplasmacytic lymphoma, splenicmargina zone lymphoma, plasma cell neoplasms, such as plasma cellmyeloma, plasmacytoma, monoclonal immunoglobulin deposition disease,heavy chain disease, MALT lymphoma, nodal marginal B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma,lymphomatoid granulomatosis, non-Hodgkins lymphoma, Hodgkins lymphoma,hairy cell leukemia, primary effusion lymphoma and AIDS-relatednon-Hodgkins lymphoma.

“Treatment” refers to the administration of an effective amount of atherapeutically active compound of the present invention with thepurpose of easing, ameliorating, arresting or eradicating (curing)symptoms or disease states.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve a desired therapeutic result. Atherapeutically effective amount of an anti-CD38 antibody may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the anti-CD38 antibody to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the antibody orantibody portion are outweighed by the therapeutically beneficialeffects.

An “anti-idiotypic” (Id) antibody is an antibody which recognizes uniquedeterminants generally associated with the antigen-binding site of anantibody.

Antibodies of the Invention

The invention relates to an antibody that binds to human CD38 (SEQ IDNO: 52), wherein the antibody does not bind to a variant of human CD38wherein Asp in position 202 has been substituted with Gly to the samedegree that it binds to human CD38. In one embodiment, the EC50 of thebinding of the antibody to the variant of human CD38 wherein Asp inposition 202 has been substituted with Gly is less than 50%, such asless than 10%, less than 5%, or less than 1% of the EC50 of the bindingof the antibody to human CD38.

In one embodiment, the antibody as defined above binds to a variant ofhuman CD38 wherein Gln in position 272 has been substituted with Arg tothe same degree that it binds to human CD38. In one embodiment, the EC50of the binding of the antibody to the variant of human CD38 wherein Glnin position 272 has been substituted with Arg is at least 80%, such asat least 90%, such as at least 95%, such as at least 98% of the EC50 ofthe binding of the antibody to human CD38.

In one embodiment, the antibody as defined in any of the embodimentsabove binds to a variant of human CD38 wherein the Ser in position 274has been substituted with Phe to the same degree that it binds to humanCD38. In one embodiment, the EC50 of the binding of the antibody to avariant of human CD38 is at least 75%, such as at least 80%, such as atleast 90%, such as at least 95%, such as at least 98% of the EC50 of thebinding of the antibody to human CD38.

In one embodiment, the antibody as defined above possesses the followingbinding characteristics: (i) it does not bind to a variant of human CD38wherein Asp in position 202 has been substituted with Gly to the samedegree that it binds to human CD38, (ii) it binds to a variant of humanCD38 wherein Gln in position 272 has been substituted with Arg to thesame degree that it binds to human CD38, (iii) it binds to a variant ofhuman CD38 wherein the Ser in position 274 has been substituted with Pheto the same degree that it binds to human CD38.

In one embodiment, the antibody as defined in any of the embodimentsabove binds human CD38 and has an inhibitory effect on the CD38 cyclaseactivity and a stimulatory effect on the CD38 hydrolase activity asmeasured in the assays of Example 8, such as wherein the inhibitoryeffect is at least 50-66% compared to the inhibitory effect on the CD38cyclase activity in the absence of antibody.

In one embodiment, the antibody as defined in any of the embodimentsabove is encoded by a human heavy chain nucleic acid comprising anucleotide sequence in its variable region as set forth in SEQ ID NO: 1,6, 11, 16 or 21, and a human light chain nucleic acid comprising anucleotide sequence in its variable region as set forth in SEQ ID NOs:26, 31, 36, 41 or 46.

In one embodiment, the antibody as defined in any of the embodimentsabove is encoded by a human heavy chain and a human light chain nucleicacid comprising nucleotide sequences in their variable regions as setforth in SEQ ID NOs: 1 and 26, 6 and 31, 11 and 36, 16 and 41, or 21 and46, respectively.

In one embodiment, the antibody as defined in any of the embodimentsabove comprises a VH CDR3 comprising

-   -   a) the sequence as set forth in SEQ ID NOs: 5, 10, 15, 20 or 25,        or    -   b) a variant of said sequence, such as a variant having at most        1, 2 or 3 amino acid modifications, preferably substitutions,        such as conservative substitutions.

In one embodiment, the antibody as defined in any of the embodimentsabove comprises a VH CDR3 having the sequence set forth in SEQ ID NOs:5, 10, 15, 20 or 25, and comprising a VL CDR3 having the sequence setforth in SEQ ID NO: 30, 35, 40, 45 or 50.

In one embodiment, the antibody as defined in any of the embodimentsabove comprises SEQ ID NO: 5 and SEQ ID NO: 30, or SEQ ID NO: 10 and SEQID NO: 35, or SEQ ID NO: 15 and SEQ ID NO: 40, or SEQ ID NO: 20 and SEQID NO: 45, or SEQ ID NO: 25 and SEQ ID NO: 50 as the VH CDR3 and VL CDR3respectively.

In one embodiment, the antibody as defined in any of the embodimentsabove comprises

(i) a VH CDR1 having the sequence as set forth in any of the sequencesSEQ ID NOs: 3, 8, 13, 18 and 23, a VH CDR2 having the sequence as setforth in any of the sequences SEQ ID NOs: 4, 9, 14, 19 and 24, a VH CDR3having the sequence as set forth in any of the sequences SEQ ID NOs: 5,10, 15, 20 and 25, a VL CDR1 having the sequence as set forth in any ofthe sequences SEQ ID NO: 28, 33, 38, 43 and 48, a VL CDR2 having thesequence as set forth in any of the sequences SEQ ID NOs: 29, 34, 39, 44and 49, a VL CDR3 having the sequence as set forth in any of thesequences SEQ ID NOs: 30, 35, 40, 45 and 50,

(ii) a VH CDR1 having the sequence as set forth in SEQ ID NO: 3, a VHCDR2 having the sequence as set forth in SEQ ID NOs: 4, a VH CDR3 havingthe sequence as set forth in SEQ ID NO: 5, a VL CDR1 having the sequenceas set forth in SEQ ID NO: 28, a VL CDR2 having the sequence as setforth in SEQ ID NO: 29, a VL CDR3 having the sequence as set forth inSEQ ID NO: 30,

(iii) a VH CDR1 having the sequence as set forth in SEQ ID NO: 8, a VHCDR2 having the sequence as set forth in SEQ ID NOs: 9, a VH CDR3 havingthe sequence as set forth in SEQ ID NO: 10, a VL CDR1 having thesequence as set forth in SEQ ID NO 33, a VL CDR2 having the sequence asset forth in SEQ ID NO: 34, a VL CDR3 having the sequence as set forthin SEQ ID NO: 35,

(iv) a VH CDR1 having the sequence as set forth in SEQ ID NO: 13, a VHCDR2 having the sequence as set forth in SEQ ID NO: 14, a VH CDR3 havingthe sequence as set forth in SEQ ID NO: 15, a VL CDR1 having thesequence as set forth in SEQ ID NO: 38, a VL CDR2 having the sequence asset forth in SEQ ID NO: 39, a VL CDR3 having the sequence as set forthin SEQ ID NO: 40,

(v) a VH CDR1 having the sequence as set forth in SEQ ID NO: 18, a VHCDR2 having the sequence as set forth in SEQ ID NOs: 19, a VH CDR3having the sequence as set forth in SEQ ID NO: 20, a VL CDR1 having thesequence as set forth in SEQ ID NO: 43, a VL CDR2 having the sequence asset forth in SEQ ID NO: 44, a VL CDR3 having the sequence as set forthin SEQ ID NO: 45,

(vi) a VH CDR1 having the sequence as set forth in SEQ ID NO: 23, a VHCDR2 having the sequence as set forth in SEQ ID NOs: 24, a VH CDR3having the sequence as set forth in SEQ ID NO: 25, a VL CDR1 having thesequence as set forth in SEQ ID NO 48, a VL CDR2 having the sequence asset forth in SEQ ID NO: 49, a VL CDR3 having the sequence as set forthin SEQ ID NO: 50, or

(vii) a variant of any of the antibodies defined above, wherein saidvariant preferably has at most 1, 2 or 3 amino acid modifications, morepreferably amino-acid substitutions, such as conservative amino acidsubstitutions in one or more of said sequences.

In one embodiment, the antibody as defined in any of the embodimentsabove comprises a VH region

(i) comprising the sequence of SEQ ID NOs: 2, 7, 12, 17 or 22, or

(ii) having at least 80% identity, such as 90%, or 95%, or 97%, or 98%,or 99% or 100% identity to the VH region sequence set forth in SEQ IDNOs: 2, 7, 12, 17 or 22.

In one embodiment, the antibody as defined in any of the embodimentsabove comprises a VL region

(i) comprising the sequence of SEQ ID NOs: 27, 32, 37, 42 or 47, or

(ii) having at least 80% identity, such as 90%, or 95%, or 97%, or 98%,or 99% or 100% identity to a VL region sequence selected from the groupconsisting of: SEQ ID NOs: 27, 32, 37, 42 or 47.

In one embodiment, the antibody as defined in any of the embodimentsabove comprises a VH region comprising any of the sequences of SEQ IDNOs: 2, 7, 12, 17 and 22, and a VL region comprising any of thesequences of SEQ ID NOs: 27, 32, 37, 42 and 47.

In one embodiment, the antibody as defined in any of the embodimentsabove comprises

(i) a VH region comprising the sequence as set forth in SEQ ID NO: 2,and a VL region comprising any the sequence as set forth in SEQ ID NO:27,

(ii) a VH region comprising the sequence as set forth in SEQ ID NO: 7,and a VL region comprising any the sequence as set forth in SEQ ID NO:32,

(iii) a VH region comprising the sequence as set forth in SEQ ID NO: 12,and a VL region comprising any the sequence as set forth in SEQ ID NO:37,

(iv) a VH region comprising the sequence as set forth in SEQ ID NO: 17,and a VL region comprising any the sequence as set forth in SEQ ID NO:42, or

(v) a VH region comprising the sequence as set forth in SEQ ID NO: 22,and a VL region comprising any the sequence as set forth in SEQ ID NO:47.

In one embodiment, the invention relates to an anti-CD38 antibody whichbinds to the same epitope on CD38 as an anti-CD38 antibody as describedin any one of the embodiments above.

In one embodiment, the invention relates to an anti-CD38 antibody whichhas substantially the same specific binding characteristics for bindinghuman CD38 as described in any one of the embodiments above.

In one embodiment, the antibody as defined in any of the embodimentsabove is capable of inducing antibody-dependent cellular cytotoxicity(ADCC), such as in Daudi cells, preferably with an EC₅₀ value of 5 nM orless, e.g. 1 nM or less, such as 0.2 nM or less, as determined by themethod described in Example 6 herein.

In one embodiment, the antibody as defined in any of the embodimentsabove is not capable of inducing ADCC in Daudi cells according to themethod described in Example 6 herein.

In one embodiment, the antibody as defined in any of the embodimentsabove is not capable of inducing complement-dependent cytotoxicity (CDC)in CHO-CD38 cells.

In one embodiment, the antibody as defined in any of the embodimentsabove binds to human CD38 with a K_(D) of 10⁻⁸ M or less, preferablywith a K_(D) of 10⁻⁹ M or less.

In one embodiment, the antibody as defined in any of the embodimentsabove is a human monovalent antibody.

In one embodiment, the antibody as defined in any of the embodimentsabove is a full length IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgMantibody, such as an IgG1 antibody, preferably an IgG1,κ antibody or anIgM antibody, preferably an IgM,κ antibody.

In one embodiment, the antibody as defined in any of the embodimentsabove is an antibody fragment or a single-chain antibody.

In one embodiment, the antibody as defined in any of the embodimentsabove is an effector-function-deficient antibody, such as a stabilizedhuman IgG4 antibody.

In one embodiment, such stabilized IgG4 antibody is an antibody whereinarginine at position 409 in the heavy chain constant region of humanIgG4 is substituted with lysine, threonine, methionine, or leucine,preferably lysine. In one embodiment, such antibody comprises a Lysresidue at the position corresponding to 409 or the CH3 region of theantibody has been replaced by the CH3 region of human IgG1, of humanIgG2 or of human IgG3. In one embodiment, such antibody does notcomprise a Cys-Pro-Pro-Cys sequence in the hinge region. In anotherembodiment, such antibody does comprise a Cys-Pro-Pro-Cys sequence inthe hinge region.

In one embodiment, the antibody as defined in any of the embodimentsabove is a monovalent antibody.

In one embodiment, such monovalent antibody is constructed by a methodcomprising:

i) providing a nucleic acid construct encoding the light chain of saidmonovalent antibody, said construct comprising a nucleotide sequenceencoding the VL region of SEQ ID NO: 27, 32, 37, 42 or 47 and anucleotide sequence encoding the constant CL region of an Ig, whereinsaid nucleotide sequence encoding the VL region of a selected antigenspecific antibody and said nucleotide sequence encoding the CL region ofan Ig are operably linked together, and wherein, in case of an IgG1subtype, the nucleotide sequence encoding the CL region has beenmodified such that the CL region does not contain any amino acidscapable of forming disulfide bonds or covalent bonds with other peptidescomprising an identical amino acid sequence of the CL region in thepresence of polyclonal human IgG or when administered to an animal orhuman being;

ii) providing a nucleic acid construct encoding the heavy chain of saidmonovalent antibody, said construct comprising a nucleotide sequenceencoding the VH region of SEQ ID NO: 2, 7, 12 17 or 22 and a nucleotidesequence encoding a constant CH region of a human Ig, wherein thenucleotide sequence encoding the CH region has been modified such thatthe region corresponding to the hinge region and, as required by the Igsubtype, other regions of the CH region, such as the CH3 region, doesnot comprise any amino acid residues which participate in the formationof disulphide bonds or covalent or stable non-covalent inter-heavy chainbonds with other peptides comprising an identical amino acid sequence ofthe CH region of the human Ig in the presence of polyclonal human IgG orwhen administered to an animal human being, wherein said nucleotidesequence encoding the VH region of a selected antigen specific antibodyand said nucleotide sequence encoding the CH region of said Ig areoperably linked together;

iii) providing a cell expression system for producing said monovalentantibody;

iv) producing said monovalent antibody by co-expressing the nucleic acidconstructs of (i) and (ii) in cells of the cell expression system of(iii).

In one embodiment, the CH region comprising the CH2 and CH3 regions hasbeen modified such that the region corresponding to the hinge regionand, if the immunoglobulin is not an IgG4 subtype, other regions of theCH region, such as the CH3 region, do not comprise any amino acidresidues, which are capable of forming disulfide bonds with an identicalCH region or other covalent or stable non-covalent inter-heavy chainbonds with an identical CH region in the presence of polyclonal humanIgG.

In one embodiment, such monovalent antibody is of the IgG4 subtype, butthe CH3 region has been modified so that one or more of the followingamino acid substitutions have been made: Thr (T) in position 366 hasbeen replaced by Ala (A); Leu (L) in position 368 has been replaced byAla (A); Leu (L) in position 368 has been replaced by Val (V); Phe (F)in position 405 has been replaced by Ala (A); Phe (F) in position 405has been replaced by Leu (L); Tyr (Y) in position 407 has been replacedby Ala (A); Arg (R) in position 409 has been replaced by Ala (A).

In one embodiment, the heavy chain of such monovalent antibody has beenmodified such that the entire hinge has been deleted.

In one embodiment, the sequence of said monovalent antibody has beenmodified so that it does not comprise any acceptor sites for N-linkedglycosylation.

In one embodiment, the antibody as defined in any of the embodimentsabove inhibits the CD38 catalyzed synthesis of cGDPR by at least 25%,such as at least 30% after 90 minutes at a concentration of 3 μg/ml asdetermined by the spectophotometric method described in Example 8 of thespecification.

In one embodiment, the antibody as defined in any of the embodimentsabove inhibits the CD38 catalyzed synthesis of cADPR by at least 25%,such as at least 30% after 90 minutes at a concentration of 3 μg/ml asdetermined by the HPLC method described in Munshi et al., 3. Biol. Chem.275, 21566-21571 (2000).

In one embodiment, the antibody stimulates the hydrolase activity ofCD38 by at least 25%.

In one embodiment, the antibody stimulates the NAD hydrolase activity ofCD38 by at least 25%.

In one embodiment, the antibody as defined in any of the embodimentsabove stimulates the cADPR-hydrolase activity of CD38 by at least 25%.

In one embodiment, the antibody as defined in any of the embodimentsabove inhibits the ability of CD38 to catalyze the formation, via abase-exchange reaction, of NAADP with an IC50 of below 0.5 μg/mL, suchas of below 0.2 μg/mL by the method described in Example 8 of thespecification.

In one embodiment, the invention relates to an antibody drug conjugatecomprising an antibody as defined in any of the embodiments above,wherein the antibody has been conjugated to a cytotoxic agent, aradioisotope, or a drug. In one embodiment, the antibody has beenconjugated to an auristatin or a functional peptide analog or derivatethereof via a linker.

In one embodiment, the invention relates to a bispecific antibodycomprising an antibody as defined in any of the embodiments above and asecond binding specificity for a human effector cell or a cancerantigen. In one embodiment, the second binding specificity is for ahuman Fc receptor or for a T cell receptor, such as CD3.

In one embodiment, the invention relates to an isolated nucleic acidencoding an antibody as defined in any of the embodiments above.

In one embodiment, the invention relates to an expression vectorcomprising a nucleotide sequence encoding one or more of the amino acidsequences as defined in any of the embodiments above.

In one embodiment, the expression vector further comprises a nucleotidesequence encoding the constant region of a light chain, a heavy chain orboth light and heavy chains of a human antibody.

In one embodiment, the invention relates to a recombinant eukaryotic orprokaryotic host cell which produces an antibody as defined in any ofthe embodiments above.

In one embodiment, the invention relates to a pharmaceutical compositioncomprising an antibody, an immunoconjugate, a bispecific antibody, or anexpression vector as defined in any of the embodiments above and apharmaceutically acceptable carrier.

In one embodiment, the invention relates to an antibody as defined inany of the embodiments above for use as a medicament.

In one embodiment, the invention relates to an antibody as defined inany of the embodiments above for use in inhibiting growth and/orproliferation, migration or inducing phagocytosis of a tumor cellexpressing CD38.

In one embodiment, the invention relates to an antibody as defined inany of the embodiments above for use in treating rheumatoid arthritis.

In one embodiment, the invention relates to an antibody as defined inany of the embodiments above for use in treating a disorder selectedfrom chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia(ALL), acute myelogenous leukemia (adults) (AML), mantle cell lymphoma,follicular lymphoma, and diffuse large B-cell lymphoma.

In one embodiment, the invention relates to an antibody as defined inany of the embodiments above for use in treating multiple myeloma.

In one embodiment, the invention relates to a method for producing ananti-CD38 antibody as defined in any of the embodiments above, saidmethod comprising the steps of

a) culturing a host cell as defined in any of the embodiments above, and

b) purifying the anti-CD38 antibody from the culture media.

In one embodiment, the invention relates to diagnostic compositioncomprising an antibody as defined in any of the embodiments above.

In one embodiment, the invention relates to a method for detecting thepresence of CD38 antigen, or a cell expressing CD38, in a samplecomprising:

-   -   contacting the sample with an anti-CD38 antibody as defined in        any of the embodiments above under conditions that allow for        formation of a complex between the antibody or bispecific        molecules and CD38; and    -   analyzing whether a complex has been formed.

In one embodiment, the invention relates to a kit for detecting thepresence of CD38 antigen, or a cell expressing CD38, in a samplecomprising an anti-CD38 antibody as defined in any of the embodimentsabove and instructions for use of the kit.

In one embodiment, the invention relates to an anti-idiotypic antibodywhich binds to an anti-CD38 antibody as defined in any of theembodiments above.

In one embodiment, the invention relates to a method of inhibitinggrowth and/or proliferation migration or inducing phagocytosis of a cellexpressing CD38, comprising administration of an antibody, animmunoconjugate, a bispecific antibody, an expression vector or apharmaceutical composition as defined in any of the embodiments above,such that the growth and/or proliferation, migration or phagocytosis ofthe cell is inhibited.

In one embodiment, the invention relates to a method of treating adisease or disorder involving cells expressing CD38 in a subject, whichmethod administration of an antibody, an immunoconjugate, a bispecificantibody, an expression vector or a pharmaceutical composition asdefined in any of the embodiments above to a subject in need thereof.

In one embodiment, the disease or disorder is rheumatoid arthritis.

In another embodiment, the disease or disorder is selected from chroniclymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (adults) (AML), mantle cell lymphoma, follicularlymphoma, and diffuse large B-cell lymphoma.

In yet another embodiment, the disease or disorder is multiple myeloma.

In one embodiment, the method as defined in any of the embodiments abovecomprises administration of one or more further therapeutic agents tothe subject, such as one or more further therapeutic agents are selectedfrom a chemotherapeutic agent, an anti-inflammatory agent, or animmunosuppressive and/or immunomodulatory agent. In one embodiment, theone or more further therapeutic agents are selected from a groupconsisting of cisplatin, gefitinib, cetuximab, rituximab, ofatumumab,bevacizumab, erlotinib, bortezomib, thalidomide, pamidronate, zoledronicacid, clodronate, risendronate, ibandronate, etidronate, alendronate,tiludronate, arsenic trioxide, lenalidomide, dexamethasone,prednisolone, filgrastim, pegfilgrastim, sargramostim, suberoylanilidehydroxamic acid, and SCIO-469.

An embodiment of the invention provides an antibody that binds to humanCD38, wherein the antibody does not bind to a variant of human CD38wherein Asp in position 202 has been substituted with Gly.

An embodiment of the invention provides an antibody according to theembodiment above, wherein the EC50 of the binding of the antibody to avariant of human CD38 is less than 50%, such as less than 10%, less than5%, or less than 1% of the EC50 of the binding of the peptide to humanCD38.

An embodiment of the invention provides an antibody according to any ofthe above embodiments, wherein the antibody binds to a variant of humanCD38 wherein the Gln in position 272 has been substituted with Arg tothe same degree that it binds to human CD38.

An embodiment of the invention provides an antibody according to theabove embodiment, wherein the EC50 of the binding of the antibody to avariant of human CD38 is at least 80%, such as at least 90%, such as atleast 95%, such as at least 98% of the EC50 of the binding of thepeptide to human CD38.

An embodiment of the invention provides an antibody according to any ofthe above embodiments, wherein the antibody binds to a variant of humanCD38 wherein the Ser in position 274 has been substituted with Phe tothe same degree that it binds to human CD38.

An embodiment of the invention provides an antibody according to theabove embodiment, wherein the EC50 of the binding of the antibody to avariant of human CD38 is at least 75%, such as at least 80%, such as atleast 90%, such as at least 95%, such as at least 98% of the EC50 of thebinding of the peptide to human CD38.

An embodiment of the invention provides an antibody according to any ofthe above embodiments, wherein the antibody possesses the followingbinding characteristics: (i) it does not bind to a variant of human CD38wherein Asp in position 202 has been substituted with Gly to the samedegree that it binds to human CD38 (ii) it binds to a variant of humanCD38 wherein the Gln in position 272 has been substituted with Arg tothe same degree that it binds to human CD38 (iii) it binds to a variantof human CD38 wherein the Ser in position 274 has been substituted withPhe to the same degree that it binds to human CD38.

An embodiment of the invention provides an antibody according to any ofthe above embodiments, that binds human CD38 and has an inhibitoryeffect on the CD38 cyclase activity and a stimulatory effect on the CD38hydrolase activity as measured in the assays of Example 8.

An embodiment of the invention provides an antibody according to theabove embodiment, wherein the inhibitory effect is at least 50-66%compared to CD38 alone.

An embodiment of the invention provides an antibody binding to humanCD38 encoded by a human heavy chain nucleic acids comprising nucleotidesequences in their variable regions as set forth the in seq id no.: 1,6, 11, 16 or 21, and a human light chain comprising nucleotide sequencesin their variable regions as set forth in seq id no. 26, 31, 36, 41 or46, and comprising conservative sequence modifications of the sequencesset forth above.

An embodiment of the invention provides an antibody according to theabove embodiment, encoded by a human heavy chain and a human light chainnucleic acids comprising nucleotide sequences in their variable regionsas set forth the in seq id no.: 1 and 26, 6 and 31, 11 and 36, 16 and 41or 21 and 46, respectively, and comprising conservative sequencemodifications of the sequences set forth above.

An embodiment of the invention provides an antibody binding to humanCD38 comprising a VH CDR3 region having

-   -   a) the sequence as set forth in SEQ ID NOs: 5, 10, 15, 20 or 25        30    -   or    -   b) a variant of said sequence, such as a variant having at most        1, 2 or 3 amino-acid modifications, preferably substitutions,        such as conservative substitutions.

An embodiment of the invention provides an antibody binding to humanCD38 comprising a VH CDR3 region having the sequence as set forth in SEQID NOs: 5, 10, 15, 20, 25 or 30 and comprising a VL CDR3 region havingthe sequence set forth in SEQ ID NO: 30, 35, 40, 45 or 50;

An embodiment of the invention provides an antibody binding to humanCD38 comprising a VH CDR3 region having the sequence as set forth in SEQID NO: 5 and a VL CDR3 region comprising SEQ ID NO: 30, or SEQ ID NO: 10and SEQ ID NO: 35, or SEQ ID NO: 15 and SEQ ID NO: 40, or SEQ ID NO: 20and SEQ ID NO: 45, or SEQ ID NO: 25 and SEQ ID NO: 45, or SEQ ID NO: 30and SEQ ID NO: 50, as VH CDR3 region and VL CDR3 region respectively.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody comprises a VHCDR1 region having the sequence as set forth in any of the sequences SEQID NOs: 3, 8, 13, 18 or 23, a VH CDR2 region having the sequence as setforth in any of the sequences SEQ ID NOs: 4, 9, 14, 19 or 24, a VL CDR3region having the sequence as set forth in any of the sequences SEQ IDNOs: 30, 35, 40, 45 or 50, and a VH CDR3 region having the sequence asset forth in SEQ ID NOs: 5, 10, 15, 20 or 25.

An embodiment of the invention provides an antibody which binds to CD38,wherein the antibody comprises a VH CDR1 region having the sequence asset forth in any of the sequences SEQ ID NOs: 3, 8, 13, 18 or 23, a VHCDR2 region having the sequence as set forth in any of the sequences SEQID NOs: 4, 9, 14, 19 or 24, a VH CDR3 region having the sequence as setforth in SEQ ID NOs: 5, 10, 15, 20 or 25, a VL CDR1 region as set forthin SEQ ID NOs: 28, 33, 38, 43 or 48, a VL CDR2 region as set forth inSEQ ID NOs: 29, 34, 39, 44 or 49, a VL CDR3 region having the sequenceas set forth in any of the sequences SEQ ID NOs: 30, 35, 40, 45 or 50 or

a variant of said antibody, wherein said variant preferably has at most1, 2 or 3 amino-acid modifications, more preferably amino-acidsubstitutions, such as conservative amino-acid substitutions in saidsequences.

An embodiment of the invention provides an antibody which binds to CD38,wherein the antibody comprises a VH CDR1 region having the sequence asset forth in any of the sequences SEQ ID NOs: 3, 8, 13, 18 or 23, a VHCDR2 region having the sequence as set forth in any of the sequences SEQID NOs: 4, 9, 14, 19 or 24, a VH CDR3 region having the sequence as setforth in SEQ ID NOs: 5, 10, 15, 20 or 25, a VL CDR1 region as set forthin SEQ ID NOs: 28, 33, 38, 43 or 48, a VL CDR2 region as set forth inSEQ ID NOs: 29, 34, 39, 44 or 49, a VL CDR3 region having the sequenceas set forth in any of the sequences SEQ ID NOs: 30, 35, 40, 45 or 50;

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, comprising a VH having at least 80%identity, such as 90%, or 95%, or 97%, or 98%, or 99% or 100% identityto the VH region sequence set forth in SEQ ID NOs: 2, 7, 12, 17, or 22.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, comprising a VL having at least 80%identity, such as 90%, or 95%, or 97%, or 98%, or 99% or 100% identityto a VL region sequence selected from the group consisting of: SEQ IDNOs: 27, 32, 37, 42 or 47.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, comprising a VH region comprising thesequence of SEQ ID NOs: 2, 7, 12, 17 or 22 and a VL region comprisingthe sequence of SEQ ID NOs: SEQ ID NOs: 27, 32, 37, 42 or 47.

according to any of the above embodiments according to any of the aboveembodiments according to any of the above embodiments

An embodiment of the invention provides an antibody which competes withan antibody according to any of the above embodiments, for binding toCD38.

An embodiment of the invention provides an anti-CD38 antibody, whichcompetes for CD38 binding with an anti-CD38 antibody comprising a VHregion comprising any of the sequences of SEQ ID NOs: 2, 7, 12, 17 or 22and a VL region comprising any of the sequences of SEQ ID NO: 27, 32,37, 42 or 47.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody binds to the sameepitope on CD38 as an anti-CD38 antibody as described in any of theabove embodiments.

An embodiment of the invention provides an antibody having substantiallythe same specific binding characteristics for binding human CD38 has anantibody according to any of the above embodiments.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody is capable ofinducing complement-dependent cytotoxicity (CDC) in CHO-CD38 cells.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody is capable ofinducing antibody-dependent cellular cytotoxicity (ADCC).

An embodiment of the invention provides an anti-CD38 antibody of claim25, wherein said antibody induces ADCC in Daudi cells, preferably withan EC₅₀ value of 5 nM or less, e.g. 1 nM or less, such as 0.2 nM orless, as determined by the method described in Example 6 herein.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody is not capable ofinducing ADCC.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody is not capable ofinducing complement-dependent cytotoxicity (CDC).

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody binds to humanCD38 with a K_(D) of 10⁻⁸ M or less, preferably with a K_(D) of 10⁻⁹ Mor less.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody comprises:

-   -   a heavy chain variable region derived from a human germline VH        sequence selected from the group consisting of: IGHV1-69*04,        and/or IGHJ3*02    -   a light chain variable region derived from a human germline Vκ        sequence selected from the group consisting of: IGKV1D-16*01,        and/or IGKJ4*01.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, which is a human antibody.

An embodiment of the invention provides an antibody according to any ofthe above embodiments, characterized in that it is a full length IgG1,IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody, such as an IgG1antibody, preferably an IgG1,κ antibody or an IgM antibody, preferablyan IgM,κ antibody.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody is an antibodyfragment or a single-chain antibody.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody is conjugated toanother moiety, such as a cytotoxic moiety, a radioisotope or a drug.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody is aneffector-function-deficient antibody.

An embodiment of the invention provides an anti-CD38 antibody of theabove embodiment, wherein the effector-function-deficient anti-CD38antibody is a stabilized human IgG4 antibody.

An embodiment of the invention provides an anti-CD38 antibody of theabove embodiment, wherein the stabilized IgG4 antibody is an antibodywherein arginine at position 409 in the heavy chain constant region ofhuman IgG4 is substituted with lysine, threonine, methionine, orleucine, preferably lysine.

An embodiment of the invention provides an anti-CD38 antibody of theabove embodiment, wherein said antibody comprises a Lys residue at theposition corresponding to 409 or the CH3 region of the antibody has beenreplaced by the CH3 region of human IgG1, of human IgG2 or of humanIgG3.

An embodiment of the invention provides an anti-CD38 antibody of theabove embodiments, wherein said antibody does not comprise aCys-Pro-Pro-Cys sequence in the hinge region.

An embodiment of the invention provides an anti-CD38 antibody of theabove embodiments, wherein said antibody does comprise a Cys-Pro-Pro-Cyssequence in the hinge region.

An embodiment of the invention provides an anti-CD38 antibody accordingto any of the above embodiments, wherein the antibody is a monovalentantibody.

An embodiment of the invention provides an anti-CD38 antibody of theabove embodiment, wherein said monovalent antibody is constructed by amethod comprising:

i) providing a nucleic acid construct encoding the light chain of saidmonovalent antibody, said construct comprising a nucleotide sequenceencoding the VL region of a selected antigen specific antibody and anucleotide sequence encoding the constant CL region of an Ig, whereinsaid nucleotide sequence encoding the VL region of a selected antigenspecific antibody and said nucleotide sequence encoding the CL region ofan Ig are operably linked together, and wherein, in case of an IgG1subtype, the nucleotide sequence encoding the CL region has beenmodified such that the CL region does not contain any amino acidscapable of forming disulfide bonds or covalent bonds with other peptidescomprising an identical amino acid sequence of the CL region in thepresence of polyclonal human IgG or when administered to an animal orhuman being;

ii) providing a nucleic acid construct encoding the heavy chain of saidmonovalent antibody, said construct comprising a nucleotide sequenceencoding the VH region of a selected antigen specific antibody and anucleotide sequence encoding a constant CH region of a human Ig, whereinthe nucleotide sequence encoding the CH region has been modified suchthat the region corresponding to the hinge region and, as required bythe Ig subtype, other regions of the CH region, such as the CH3 region,does not comprise any amino acid residues which participate in theformation of disulphide bonds or covalent or stable non-covalentinter-heavy chain bonds with other peptides comprising an identicalamino acid sequence of the CH region of the human Ig in the presence ofpolyclonal human IgG or when administered to an animal human being,wherein said nucleotide sequence encoding the VH region of a selectedantigen specific antibody and said nucleotide sequence encoding the CHregion of said Ig are operably linked together;

iii) providing a cell expression system for producing said monovalentantibody;

iv) producing said monovalent antibody by co-expressing the nucleic acidconstructs of (i) and (ii) in cells of the cell expression system of(iii).

An embodiment of the invention provides an anti-CD38 antibody of theabove embodiment, wherein the monovalent antibody comprises

(i) a variable region of an antibody according to any of the aboveembodiments, or an antigen binding part of the said region, and

(ii) a C_(H) region of an immunoglobulin or a fragment thereofcomprising the C_(H)2 and C_(H)3 regions, wherein the C_(H) region orfragment thereof has been modified such that the region corresponding tothe hinge region and, if the immunoglobulin is not an IgG4 subtype,other regions of the C_(H) region, such as the CH3 region, do notcomprise any amino acid residues, which are capable of forming disulfidebonds with an identical C_(H) region or other covalent or stablenon-covalent inter-heavy chain bonds with an identical C_(H) region inthe presence of polyclonal human IgG.

An embodiment of the invention provides an anti-CD38 antibody of theabove embodiments wherein said monovalent antibody is of the IgG4subtype, but the C_(H)3 region has been modified so that one or more ofthe following amino acid substitutions have been made: Thr (T) inposition 366 has been replaced by Ala (A); Leu (L) in position 368 hasbeen replaced by Ala (A); Leu (L) in position 368 has been replaced byVal (V); Phe (F) in position 405 has been replaced by Ala (A); Phe (F)in position 405 has been replaced by Leu (L); Tyr (Y) in position 407has been replaced by Ala (A); Arg (R) in position 409 has been replacedby Ala (A).

An embodiment of the invention provides an anti-CD38 antibody of any ofthe above embodiments, the heavy chain has been modified such that theentire hinge has been deleted.

An embodiment of the invention provides an anti-CD38 antibody of any ofthe above embodiments, wherein the sequence of said monovalent antibodyhas been modified so that it does not comprise any acceptor sites forN-linked glycosylation.

An embodiment of the invention provides an antibody according to any ofthe above embodiments, which inhibits the synthesis of cGDPR by at least25%, such as at least 30% after 90 minutes as determined byspectophotometric method described in Example 8 of the specification.

An embodiment of the invention provides an antibody according to any ofthe above embodiments which inhibits the synthesis of cADPR by at least25%, such as at least 30% after 90 minutes at a concentration of 3 μg/mlas determined by the HPLC method described in Munshi et al., J. Biol.Chem. 275, 21566-21571 (2000).

An embodiment of the invention provides an antibody according to any ofthe above embodiments, which stimulate the hydrolase activity of CD38 byat least 25%.

An embodiment of the invention provides an antibody according to any ofthe above embodiments, which stimulate the NAD hydrolase activity by atleast 25%.

An embodiment of the invention provides an antibody according to any ofthe above embodiments, which stimulate the cADPR-hydrolase activity byat least 25%.

An embodiment of the invention provides an isolated nucleic acidencoding a peptide according to the any of the above embodiments.

An embodiment of the invention provides an expression vector comprisinga nucleotide sequence encoding one or more of the amino acid sequencesaccording to any of the above embodiments.

An embodiment of the invention provides an expression vector accordingto the above embodiment, further comprising a nucleotide sequenceencoding the constant region of a light chain, a heavy chain or bothlight and heavy chains of a human antibody.

An embodiment of the invention provides a recombinant eukaryotic orprokaryotic host cell which produces an antibody as defined in any ofthe above embodiments.

An embodiment of the invention provides a hybridoma which produces anantibody as defined in any of the above embodiments.

An embodiment of the invention provides a pharmaceutical compositioncomprising an antibody as defined in any of the above embodiments, and apharmaceutically acceptable carrier.

An embodiment of the invention provides an antibody as defined in any ofthe embodiments above for use as a medicament.

An embodiment of the invention provides an antibody as defined in any ofthe embodiments above for use in inhibiting growth and/or proliferation,migration or inducing phagocytosis of a tumor cell expressing CD38.

An embodiment of the invention provides an antibody as defined in any ofthe embodiments above for use in treating rheumatoid arthritis.

An embodiment of the invention provides an antibody as defined in any ofthe embodiments above for use in treating multiple myeloma.

An embodiment of the invention provides an antibody as defined in any ofthe embodiments above for use in treating multiple sclerosis.

An embodiment of the invention provides an antibody as defined in any ofthe embodiments above for use in treating B-cell neoplasms such as anyone of the following: small lymphocytic lymphoma, B-cell prolymphocyticlymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma,Burkitt's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,multiple myeloma, lymphoplasmacytic lymphoma, splenic margina zonelymphoma, plasma cell neoplasms, such as plasma cell myeloma,plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chaindisease, MALT lymphoma, nodal marginal B cell lymphoma, intravascularlarge B cell lymphoma, primary effusion lymphoma, lymphomatoidgranulomatosis, non-Hodgkins lymphoma, Hodgkins lymphoma, hairy cellleukemia, primary effusion lymphoma or AIDS-related non-Hodgkinslymphoma

An embodiment of the invention provides a method for inhibiting growthand/or proliferation, migration or inducing phagocytosis of a tumor cellexpressing CD38, comprising administration, to an individual in needthereof, of an antibody of any of the above embodiments.

An embodiment of the invention provides a method for producing ananti-CD38 antibody of any of the above embodiments, said methodcomprising the steps of

a) culturing a host cell of claim 52 or a hybridoma of the aboveembodiment, and

b) purifying the anti-CD38 antibody from the culture media.

An embodiment of the invention provides a diagnostic compositioncomprising an antibody as defined in any of the above embodiments.

An embodiment of the invention provides a method for detecting thepresence of CD38 antigen, or a cell expressing CD38, in a samplecomprising:

-   -   contacting the sample with an anti-CD38 antibody of any of the        above embodiments under conditions that allow for formation of a        complex between the antibody or bispecific molecules and CD38;        and    -   analyzing whether a complex has been formed.

An embodiment of the invention provides a kit for detecting the presenceof CD38 antigen, or a cell expressing CD38, in a sample comprising

-   -   an anti-CD38 antibody of any of the above embodiments or        and    -   instructions for use of the kit.

An embodiment of the invention provides an anti-idiotypic antibody whichbinds to an anti-CD38 antibody of any of the above embodiments.

An embodiment of the invention provides a method of inhibiting growthand/or proliferation of a cell expressing CD38, comprisingadministration of a peptide according to any of the above embodiments,an immunoconjugate according to the above embodiment, a pharmaceuticalcomposition according to the above embodiments or an expression vectormentioned in the above embodiments, such that the growth and/orproliferation, migration or phagocytosis of the cell is inhibited.

An embodiment of the invention provides a method of treating a diseaseor disorder involving cells expressing CD38 in a subject, which methodcomprises administration of a peptide according to any of the aboveembodiments, an immunoconjugate according to an embodiment above, apharmaceutical composition according to an embodiment above, or anexpression vector according to any one the embodiments above to asubject in need thereof.

An embodiment of the invention provides a method of preventing a diseaseor disorder involving cells expressing CD38 in a subject, which methodcomprises administration of a peptide according to any of the aboveembodiments, an immunoconjugate according to an embodiment above, apharmaceutical composition according to an embodiment above, or anexpression vector according to any one the embodiments above to asubject in need thereof.

An embodiment of the invention provides a method according to the aboveembodiments wherein the disease or disorder is rheumatoid arthritis.

An embodiment of the invention provides a method according to the aboveembodiments wherein the disease or disorder is B-cell neoplasms such asany one of the following: small lymphocytic lymphoma, B-cellprolymphocytic lymphoma, B-cell chronic lymphocytic leukemia, mantlecell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse largeB-cell lymphoma, multiple myeloma, lymphoplasmacytic lymphoma, splenicmargina zone lymphoma, plasma cell neoplasms, such as plasma cellmyeloma, plasmacytoma, monoclonal immunoglobulin deposition disease,heavy chain disease, MALT lymphoma, nodal marginal B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma,lymphomatoid granulomatosis, non-Hodgkins lymphoma, Hodgkins lymphoma,hairy cell leukemia, primary effusion lymphoma and AIDS-relatednon-Hodgkins lymphoma.

An embodiment of the invention provides a method according to the aboveembodiments wherein the disease or disorder is multiple myeloma Anembodiment of the invention provides a method according to the aboveembodiments wherein the disease or disorder is autoimmune disease.

An embodiment of the invention provides a method according to the aboveembodiments wherein the disease or disorder is diabetes.

An embodiment of the invention provides a method according to the aboveembodiments wherein the disease or disorder is multiple sclerosis.

An embodiment of the invention provides a method according to the aboveembodiments wherein the disease or disorder is Grave's disease.

An embodiment of the invention provides a method according to the aboveembodiments wherein the disease or disorder is neuroinflammation.

An embodiment of the invention provides a method according to the aboveembodiments wherein the disease or disorder is inflammation of airwaysmooth muscle cells during asthma.

An embodiment of the invention provides a method according to the aboveembodiments, wherein the method comprises administration of one or morefurther therapeutic agents to the subject.

An embodiment of the invention provides a method according to the aboveembodiment, wherein the one or more further therapeutic agents areselected from a chemotherapeutic agent, an anti-inflammatory agent, oran immunosuppressive and/or immunomodulatory agent.

An embodiment of the invention provides a method according to the aboveembodiment, wherein the one or more further therapeutic agents areselected from a group consisting of cisplatin, gefitinib, cetuximab,rituximab, bevacizumab, erlotinib, bortezomib, thalidomide, pamidronate,zoledronic acid, clodronate, risendronate, ibandronate, etidronate,alendronate, tiludronate, arsenic trioxide, lenalidomide, filgrastim,pegfilgrastim, sargramostim, suberoylanilide hydroxamic acid, andSCIO-469.

Monoclonal antibodies of the present invention may e.g. be produced bythe hybridoma method first described by Kohler et al., Nature 256, 495(1975), or may be produced by recombinant DNA methods. Monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in, for example, Clackson et al., Nature 352,624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991).Monoclonal antibodies may be obtained from any suitable source. Thus,for example, monoclonal antibodies may be obtained from hybridomasprepared from murine splenic B cells obtained from mice immunized withan antigen of interest, for instance in form of cells expressing theantigen on the surface, or a nucleic acid encoding an antigen ofinterest.

In one embodiment, the antibody of the invention is a human antibody.Human monoclonal antibodies directed against CD38 may be generated usingtransgenic or transchromosomal mice carrying parts of the human immunesystem rather than the mouse system. Such transgenic andtranschromosomic mice include mice referred to herein as HuMAb mice andKM mice, respectively, and are collectively referred to herein as“transgenic mice”.

The HuMAb mouse contains a human immunoglobulin gene miniloci thatencodes unrearranged human heavy (μ and γ) and κ light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature368, 856-859 (1994)). Accordingly, the mice exhibit reduced expressionof mouse IgM or κ and in response to immunization, the introduced humanheavy and light chain transgenes, undergo class switching and somaticmutation to generate high affinity human IgG,κ monoclonal antibodies(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook ofExperimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar,D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. andLonberg, N. Ann. N.Y. Acad. Sci 764 536-546 (1995)). The preparation ofHuMAb mice is described in detail in Taylor, L. et al., Nucleic AcidsResearch 20, 6287-6295 (1992), Chen, J. et al., International Immunology5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994),Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild,D. et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat.Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397,5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.

The HCo7 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429).

The HCo12 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo12 human heavy chain transgene (as described in Example 2 of WO01/14424).

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according to wellknown techniques.

Human monoclonal or polyclonal antibodies of the present invention, orantibodies of the present invention originating from other species mayalso be generated transgenically through the generation of anothernon-human mammal or plant that is transgenic for the immunoglobulinheavy and light chain sequences of interest and production of theantibody in a recoverable form therefrom. In connection with thetransgenic production in mammals, antibodies may be produced in, andrecovered from, the milk of goats, cows, or other mammals. See forinstance U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172 and 5,741,957.

Further, human antibodies of the present invention or antibodies of thepresent invention from other species may be generated and identifiedthrough display-type technologies, including, without limitation, phagedisplay, retroviral display, ribosomal display, and other techniques,using techniques well known in the art and the resulting molecules maybe subjected to additional maturation, such as affinity maturation, assuch techniques are well known in the art (see for instance Hoogenboomet al., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al.,Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNASUSA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992),Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. AcidsResearch 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130,43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and U.S.Pat. No. 5,733,743). If display technologies are utilized to produceantibodies that are not human, such antibodies may be humanized.

Competition for binding to CD38 or a portion of CD38 by two or moreanti-CD38 antibodies may be determined by any suitable technique.Competition in the context of the present invention refers to anydetectably significant reduction in the propensity for a particularmolecule to bind a particular binding partner in the presence of anothermolecule that binds the binding partner. Typically, competition means anat least about 10% reduction, such as an at least about 15%, or an atleast about 20% reduction in binding between an anti-CD38 antibody and

-   -   (a) a form of CD38 (e.g. “processed”, “mature”, “unprocessed”,        “not processed” or “immature” CD38);    -   (b) a form of free CD38 (e.g., a CD38 fragment produced by in        vivo processing);    -   (c) a heterodimeric peptide composed of another peptide        associated with CD38, such as CD31 associated with CD38;    -   (d) a complex of CD38 and one or more substrates, such as cAMP,        NAD+ and/or cADPR;    -   (e) a dimerized, associated and/or processed dimer of CD38 with        a soluble ligand, such as CD31; or    -   (f) a portion of CD38,        caused by the presence of another anti-CD38 antibody as        determined by, e.g., ELISA analysis or FACS analysis (as        described in the examples section) using sufficient amounts of        the two or more competing anti-CD38 antibodies and CD38        molecule. It may also be the case that competition may exist        between anti-CD38 antibodies with respect to more than one form        of CD38, and/or a portion of CD38, e.g. in a context where the        antibody-binding properties of a particular region of CD38 are        retained in fragments thereof, such as in the case of a        well-presented linear epitope located in various tested        fragments or a conformational epitope that is presented in        sufficiently large CD38 fragments as well as in CD38.

Assessing competition typically involves an evaluation of relativeinhibitory binding using a first amount of a first molecule; a secondamount of a second molecule; and a third amount of a third molecule (ora standard determined by binding studies that may be reasonably comparedto new binding data with respect to the first and second molecules as asurrogate for actual contemporaneous data), wherein the first, second,and third amounts all are sufficient to make a comparison that impartsinformation about the selectivity and/or specificity of the molecules atissue with respect to the other present molecules. The first, second,and third amounts may vary with the nature of the anti-CD38 antibody andpotential targets therefore at issue. For instance, for ELISAassessments, similar to those described in the Examples section, about5-50 μg (e.g., about 10-50 μg, about 20-50 μg, about 5-20 μg, about10-20 μg, etc.) of anti-CD38 antibody and/or CD38 targets are requiredto assess whether competition exists. Conditions also should be suitablefor binding. Typically, physiological or near-physiological conditions(e.g., temperatures of about 20-40° C., pH of about 7-8, etc.) aresuitable for anti-CD38 antibody:CD38 binding. Often competition ismarked by a significantly greater relative inhibition than about 5% asdetermined by ELISA and/or FACS analysis. It may be desirable to set ahigher threshold of relative inhibition as a criteria/determinant ofwhat is a suitable level of competition in a particular context (e.g.,where the competition analysis is used to select or screen for newantibodies designed with the intended function of blocking the bindingof another peptide or molecule binding to CD38 (e.g., the naturalbinding partners of CD38 such as CD31, also called CD31 antigen,EndoCAM, GPIIA′, PECAM-1, platelet/endothelial cell adhesion molecule ornaturally occurring anti-CD38 antibody)). Thus, for example, it ispossible to set a criterion for competitiveness wherein at least about10% relative inhibition is detected; at least about 15% relativeinhibition is detected; or at least about 20% relative inhibition isdetected before an antibody is considered sufficiently competitive. Incases where epitopes belonging to competing antibodies are closelylocated in an antigen, competition may be marked by greater than about40% relative inhibition of CD38 binding (e.g., at least about 45%inhibition, such as at least about 50% inhibition, for instance at leastabout 55% inhibition, such as at least about 60% inhibition, forinstance at least about 65% inhibition, such as at least about 70%inhibition, for instance at least about 75% inhibition, such as at leastabout 80% inhibition, for instance at least about 85% inhibition, suchas at least about 90% inhibition, for instance at least about 95%inhibition, or higher level of relative inhibition).

Competition may be considered the inverse of cross-reactivity between amolecule and two potential binding partners. In certain embodiments, aanti-CD38 antibody of the present invention specifically binds to one ormore residues or regions in CD38 but also does not cross-react withother peptides, peptide regions, or molecules, e.g., the presentinvention provides an anti-CD38 antibody that does not cross-react withproteins with homology to CD38, such as BST-1 (bone marrow stromal cellantigen-1) and Mo5, also called CD157; or anti-CD38 antibodies that donot cross-react with CD38 in the context of normal tissue, such astissues not involved in multiple myeloma. Typically, a lack ofcross-reactivity means less than about 5% relative competitiveinhibition between the molecules when assessed by ELISA and/or FACSanalysis using sufficient amounts of the molecules under suitable assayconditions.

In one embodiment, the present invention provides an anti-CD38 antibodythat competes with an antibody having a V_(L) sequence of SEQ ID NO: 27and a V_(H) sequence of SEQ ID NO: 2, such as the antibody 028, forbinding to CD38 or a portion thereof.

In one embodiment, the present invention provides an anti-CD38 antibodythat competes with an antibody having a V_(L) sequence of SEQ ID NO: 32and a V_(H) sequence of SEQ ID NO: 7, such as the antibody 025, forbinding to CD38 or a portion thereof.

In one embodiment, the present invention provides an anti-CD38 antibodythat competes with an antibody having a V_(L) sequence of SEQ ID NO: 37and a V_(H) sequence of SEQ ID NO: 12, such as the antibody 026, forbinding to CD38 or a portion thereof.

In one embodiment, the present invention provides an anti-CD38 antibodythat competes with an antibody having a V_(L) sequence of SEQ ID NO: 42and a V_(H) sequence of SEQ ID NO: 17, such as the antibody 049, forbinding to CD38 or a portion thereof.

In one embodiment, the present invention provides an anti-CD38 antibodythat competes with an antibody having a V_(L) sequence of SEQ ID NO: 47and a V_(H) sequence of SEQ ID NO: 22, such as the antibody 056, forbinding to CD38 or a portion thereof.

As discussed elsewhere herein, unless otherwise stated or clearlycontradicted by context, references to binding of an anti-CD38 antibodyto CD38 are intended to refer to binding in any suitable context, suchas in a conformational context where the structure of CD38 is present;or in a linear epitope context. Of course, binding in a limited subsetof such context(s) may be an important characteristic with respect toany anti-CD38 antibody provided by the present invention.

Additional methods for determining anti-CD38 antibody specificity bycompetitive inhibition may be found in for instance Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988), Colligan et al., eds., CurrentProtocols in Immunology, Greene Publishing Assoc. and Wiley InterScienceN.Y., (1992, 1993), and Muller, Meth. Enzymol. 92, 589-601 (1983)).

Human CD38 comprises a number of different epitopes, which may include(1) peptide antigenic determinants that are comprised within singlepeptide chains within human CD38; (2) conformational antigenicdeterminants which consist of one or more noncontiguous amino acids on aparticular chain and/or amino acids present on spatially contiguous butseparate peptide chains (typically where the respective amino acidsequences of the chains are located disjointedly along the human CD38polypeptide sequence); (3) post-translational antigenic determinantswhich consist, either in whole or part, of molecular structurescovalently attached to human CD38, such as carbohydrate groups; or (4)combinations of (1)-(3).

An epitope in the context of the present invention includes any peptideor peptide-derivative determinant capable of specific binding to animmunoglobulin. An epitope may comprise any suitable number of aminoacids, in any suitable position (with respect to the linear sequence ofCD38), orientation (with respect to folded CD38, or a fragment thereof),amino acid composition (and consequently, at least in part, charge).Thus, for example, an epitope may be composed of about 3-10 amino acids,typically 3-8 amino acids, in one or more contiguous or noncontiguouslocations with respect to the primary sequence of CD38 (for instance anepitope may consist essentially of 2, 3, 4, 5, 6, 7, or 8 amino acidresidues distributed in 1, 2, 3, 4, or 5 noncontiguous locations inCD38). Alternatively, for example, an epitope may be considered to bedefined by a region of about 5-40 contiguous amino acid residues (e.g.,about 7-30 amino acid residues, about 5-20 amino acid residues, or about3-15 amino acid residues) in CD38 (solely or in combination with aportion of an adjacent CD38 domain). In some epitopes it may be the casethat just one amino acid residue or only a few amino acid residues arecritical to CDR or CDR(s) recognition (and thereby most important toanti-CD38 antibody:CD38 antigen affinity and avidity). As such, anepitope may be characterized on the basis of one or more of suchcritical residues, with the recognition that other residues may alsomake some lesser contribution to the epitope. In the case of an epitopedefined by a region of amino acids, it may be that one or more aminoacids in the region make only a minor contribution or even negligiblecontribution to antibody binding, such that the residue may be subjectto substitution with an appropriate different residue without resultingin “a loss” of the epitope to at least some anti-CD38 antibodiesspecific for it.

In one embodiment, the present invention provides a anti-CD38 antibody,such as an anti-CD38 antibody, that specifically binds to a CD38 epitopethat also is specifically bound by an antibody having a V_(L) sequenceof SEQ ID NO: 27 and a V_(H) sequence of SEQ ID NO: 2 (such as antibody028), or an antibody having a V_(L) sequence of SEQ ID NO: 32 and aV_(H) sequence of SEQ ID NO: 7 (such as antibody 025), or an antibodyhaving a V_(L) sequence of SEQ ID NO: 37 and a V_(H) sequence of SEQ IDNO: 12 (such as antibody 026), or an antibody having a V_(L) sequence ofSEQ ID NO: 42 and a V_(H) sequence of SEQ ID NO: 17 (such as antibody049), or an antibody having a V_(L) sequence of SEQ ID NO: 47 and aV_(H) sequence of SEQ ID NO: 22 (such as antibody 056).

It is possible that anti-CD38 antibodies having one or more CDRs thatdiffer from the CDRs of an antibody having a V_(L) sequence of SEQ IDNO: 27 and a V_(H) sequence of SEQ ID NO: 2, or the CDRs of an antibodyhaving a V_(L) sequence of SEQ ID NO: 32 and a V_(H) sequence of SEQ IDNO: 7, or the CDRs of an antibody having a V_(L) sequence of SEQ ID NO:37 and a V_(H) sequence of SEQ ID NO: 12, or the CDRs of an antibodyhaving a V_(L) sequence of SEQ ID NO: 42 and a V_(H) sequence of SEQ IDNO: 17, or the CDRs of an antibody having a V_(L) sequence of SEQ ID NO:47 and a V_(H) sequence of SEQ ID NO: 22, may still be specific for thesame epitope as an antibody having the CDRs of an antibody having aV_(L) sequence of SEQ ID NO: 27 and a V_(H) sequence of SEQ ID NO: 2, orthe CDRs of an antibody having a V_(L) sequence of SEQ ID NO: 32 and aV_(H) sequence of SEQ ID NO: 7, or the CDRs of an antibody having aV_(L) sequence of SEQ ID NO: 37 and a V_(H) sequence of SEQ ID NO: 12,or the CDRs of an antibody having a V_(L) sequence of SEQ ID NO: 42 anda V_(H) sequence of SEQ ID NO: 17, or the CDRs of an antibody having aV_(L) sequence of SEQ ID NO: 47 and a V_(H) sequence of SEQ ID NO: 22,respectively. In such cases, the anti-CD38 antibody in question mayrecognize or be more specific/selective for particular structures orregions of the epitope than the antibody having the CDRs of an antibodyhaving a V_(L) sequence of SEQ ID NO: 27 and a V_(H) sequence of SEQ IDNO: 2, or the CDRs of an antibody having a V_(L) sequence of SEQ ID NO:32 and a V_(H) sequence of SEQ ID NO: 7, or the CDRs of an antibodyhaving a V_(L) sequence of SEQ ID NO: 37 and a V_(H) sequence of SEQ IDNO: 12, or the CDRs of an antibody having a V_(L) sequence of SEQ ID NO:42 and a V_(H) sequence of SEQ ID NO: 17, or the CDRs of an antibodyhaving a V_(L) sequence of SEQ ID NO: 47 and a V_(H) sequence of SEQ IDNO: 22 respectively.

A CD38 epitope bound by an antibody having a V_(L) sequence of SEQ IDNO: 27 and a V_(H) sequence of SEQ ID NO: 2 (such as antibody 028), oran antibody having a V_(L) sequence of SEQ ID NO: 32 and a V_(H)sequence of SEQ ID NO: 7 (such as antibody 025), or an antibody having aV_(L) sequence of SEQ ID NO: 37 and a V_(H) sequence of SEQ ID NO: 12(such as antibody 026), or an antibody having a V_(L) sequence of SEQ IDNO: 42 and a V_(H) sequence of SEQ ID NO: 17 (such as antibody 049), oran antibody having a V_(L) sequence of SEQ ID NO: 47 and a V_(H)sequence of SEQ ID NO: 22 (such as antibody 056), may be identified viastandard mapping and characterization techniques, further refinement ofwhich may be identified by any suitable technique, numerous examples ofwhich are available to the skilled artisan.

These techniques may also be used to identify and/or characterizeepitopes for anti-CD38 antibodies generally. As one example of suchmapping/characterization methods, an epitope for an anti-CD38 antibodymay be determined by epitope “foot-printing” using chemical modificationof the exposed amines/carboxyls in the CD38 protein. One specificexample of such a foot-printing technique is the use of HXMS(hydrogen-deuterium exchange detected by mass spectrometry) wherein ahydrogen/deuterium exchange of receptor and ligand protein amideprotons, binding, and back exchange occurs, wherein the backbone amidegroups participating in protein binding are protected from back exchangeand therefore will remain deuterated. Relevant regions may be identifiedat this point by peptic proteolysis, fast microbore high-performanceliquid chromatography separation, and/or electrospray ionization massspectrometry. See, e.g., Ehring H, Analytical Biochemistry, 267(2)252-259 (1999) and/or Engen, J. R. and Smith, D. L. (2001) Anal. Chem.73, 256A-265A. Another example of a suitable epitope identificationtechnique is nuclear magnetic resonance epitope mapping (NMR), wheretypically the position of the signals in two-dimensional NMR spectres ofthe free antigen and the antigen complexed with the antigen bindingpeptide, such as an antibody, are compared. The antigen typically isselectively isotopically labeled with ¹⁵N so that only signalscorresponding to the antigen and no signals from the antigen bindingpeptide are seen in the NMR-spectrum. Antigen signals originating fromamino acids involved in the interaction with the antigen binding peptidetypically will shift position in the spectres of the complex compared tothe spectres of the free antigen, and the amino acids involved in thebinding may be identified that way. See for instance Ernst Schering ResFound Workshop. (44), 149-67 (2004), Huang et al., Journal of MolecularBiology 281(1), 61-67 (1998) and Saito and Patterson, Methods. 9(3),516-24 (1996).

Epitope mapping/characterization may also be performed using massspectrometry methods. See for instance Downward, J Mass Spectrom. 35(4),493-503 (2000) and Kiselar and Downard, Anal Chem. 71(9), 1792-801(1999).

Protease digestion techniques may also be useful in the context ofepitope mapping and identification. Antigenic determinant-relevantregions/sequences may be determined by protease digestion, e.g. by usingtrypsin in a ratio of about 1:50 to CD38 overnight (0/N) digestion at37° C. and pH 7-8, followed by mass spectrometry (MS) analysis forpeptide identification. The peptides protected from trypsin cleavage bythe CD38BP may subsequently be identified by comparison of samplessubjected to trypsin digestion and samples incubated with CD38BP andthen subjected to digestion by e.g. trypsin (thereby revealing afoot-print for the binder). Other enzymes like chymotrypsin, pepsin,etc. may also or alternatively be used in a similar epitopecharacterization method.

An anti-CD38 antibody which gives the significantly same result as anantibody having a V_(L) sequence of SEQ ID NO: 27 and a V_(H) sequenceof SEQ ID NO: 2 (such as antibody 028), or an antibody having a V_(L)sequence of SEQ ID NO: 32 and a V_(H) sequence of SEQ ID NO: 7 (such asantibody 025), or an antibody having a V_(L) sequence of SEQ ID NO: 37and a V_(H) sequence of SEQ ID NO: 12 (such as antibody 026), or anantibody having a V_(L) sequence of SEQ ID NO: 42 and a V_(H) sequenceof SEQ ID NO: 17 (such as antibody 049), or an antibody having a V_(L)sequence of SEQ ID NO: 47 and a V_(H) sequence of SEQ ID NO: 22 (such asantibody 056), in these measurements are deemed to be an antibody thatbind the same epitope as an antibody having a V_(L) sequence of SEQ IDNO: 27 and a V_(H) sequence of SEQ ID NO: 2 (such as antibody 028), oran antibody having a V_(L) sequence of SEQ ID NO: 32 and a V_(H)sequence of SEQ ID NO: 7 (such as antibody 025), or an antibody having aV_(L) sequence of SEQ ID NO: 37 and a V_(H) sequence of SEQ ID NO: 12(such as antibody 026), or an antibody having a V_(L) sequence of SEQ IDNO: 42 and a V_(H) sequence of SEQ ID NO: 17 (such as antibody 049), oran antibody having a V_(L) sequence of SEQ ID NO: 47 and a V_(H)sequence of SEQ ID NO: 22 (such as antibody 056), respectively. See forinstance Manca, Ann Ist Super Sanita. 27(1), 15-9 (1991) for adiscussion of similar techniques.

Epitope mapping by competitive binding to CD38 with two antibodies whereone is biotinylated is another method for identifying relevant antigenicdeterminant regions. The binding of antibodies to linear and loopedpeptides of CD38 by a PEPSCAN-based enzyme-linked immuno assay isanother method for identifying relevant antigenic determinant regions,see for instance Slootstra-J W et al. Mol-Divers. 1, 87-96 (1996).

Site directed mutagenesis is another method for identifying relevantantigenic determinant regions, see for instance Polyak and Deans, Blood99, 3956-3962 (2002). Various phage display techniques may also be usedto identify epitopes. See for instance Wang and Yu, Curr Drug Targets.5(1), 1-15 (2004), Burton, Immunotechnology. 1(2), 87-94 (1995 August),Cortese et al., Immunotechnology. 1(2), 87-94 (1995) and Irving et al.,Curr Opin Chem Biol. 5(3), 314-24 (2001). Consensus epitopes may also beidentified through modified phage display-related techniques (see,http://www.cs.montana.edu/˜mumey/papers/jcb03.pdf) for discussion.

Other methods potentially helpful in mapping epitopes includecrystallography techniques, X-ray diffraction techniques (such as theX-ray diffraction/sequence study techniques developed by Poljak andothers in the 1970s-1980s), and the application of Multipin PeptideSynthesis Technology. Computer-based methods such as sequence analysisand three dimensional structure analysis and docking may also be used toidentify antigenic determinants. For example, an epitope may also bedetermined by molecular modeling using a structure of CD38 with dockingof the structure of the Fab fragment of the individual monoclonalantibody. These and other mapping methods are discussed in EpitopeMapping A Practical Approach (Westwood and Hay Eds.) 2001 OxfordUniversity Press.

In one embodiment, the present invention provides an anti-CD38 antibodyhaving substantially the same specific CD38-binding characteristics ofone or more mAbs selected from an antibody having a V_(L) sequence ofSEQ ID NO: 27 and a V_(H) sequence of SEQ ID NO: 2 (such as antibody028), or an antibody having a V_(L) sequence of SEQ ID NO: 32 and aV_(H) sequence of SEQ ID NO: 7 (such as antibody 025), or an antibodyhaving a V_(L) sequence of SEQ ID NO: 37 and a V_(H) sequence of SEQ IDNO: 12 (such as antibody 026), or an antibody having a V_(L) sequence ofSEQ ID NO: 42 and a V_(H) sequence of SEQ ID NO: 17 (such as antibody049), or an antibody having a V_(L) sequence of SEQ ID NO: 47 and aV_(H) sequence of SEQ ID NO: 22 (such as antibody 056).

Mapping studies have indicated that several monoclonal antibodies raisedagainst human CD38 bind to epitopes in the C-terminal region of CD38(220-296) (Hoshino et al. and Ferrero et al.). Within this region threeamino acid differences have been found between the human and thecynomolgus CD38 sequence: T237, Q272 and S274 in humans correspond toA238, R273 and F275 in cynomolgus. A limited number of amino aciddifferences exist between the human and the monkey CD38 sequence, forinstance in the carboxyterminal part to the protein, for instance thefollowing three amino acid differences between the human and thecynomolgus CD38 sequence: T237, Q272 and S274 in human CD38s correspondto A238, R273 and F275 in cynomolgus monkey CD38 (compare SEQ ID No.21and SEQ ID No.22).

The antibodies of the present invention do not bind to human CD38mutants wherein aspartic acid in position 202 has been substituted witha glycine to the same degree that it binds to human CD38. The presentinvention provides antibodies, which bind to human CD38 and which bindsto a mutant human CD38, wherein the serine residue in position 274 hasbeen substituted with a phenylalanine residue. The antibodies of thepresent invention also bind to human CD38 mutants wherein glutamine inposition 272 has been substituted with an arginine. The antibodies ofthe present invention also bind to human CD38 mutants wherein thethreonine in position 237 has been substituted with an alanine.

The term “do not bind to the same degree” should be interpreted so thatthe binding of the antibody to the mutant human CD38 is significantlylower than the binding of the antibody to the wild type human CD38. Theterm “bind to the same degree” should be interpreted so that the bindingof the antibody to the mutant human CD38 is substantially of the sameorder as the binding of the antibody to the wild type human CD38. Thebinding of a peptide to the CD38 molecules (wild type and mutant) may bedetermined in a number of ways and it is within the common generalknowledge of a person skilled in the art to determine whether thebinding to the mutant is “significantly lower” than the binding to thewild type. A large number of different techniques for determining thebinding of a peptide to another peptide are available to the personskilled in the art, for example ELISA, radioimmunoassay, BIAcore or flowcytometry.

One method of determining the binding is by determining the EC₅₀ of thebinding of the antibody to the mutant protein and to the wild typeprotein and then comparing the values obtained. Another method ofdetermining the binding is by examining the magnitude of binding atsaturating concentration (for instance the plateau of binding signal),or by determining kinetic rate constants k_(on) and k_(off) for exampleby BIAcore.

In one embodiment, the binding of the antibody in question to the CD38proteins (mutant or wild type) is by use of an ELISA as described inExample 4.

In a further embodiment, the antibody of the invention comprises a humanheavy chain variable region (VH) CDR3 sequence comprising:

-   -   an amino acid sequence selected from the group consisting of:        SEQ ID NOs: 5, 10, 15, 20 and 25, or    -   a variant of any of said sequences, wherein said variant        preferably only has conservative amino acid modifications.

In one embodiment, said variant consists essentially of a sequencehaving at least about 50%, such as at least 60%, for instance at leastabout 70%, such as at least about 75%, for instance at least about 80%,such as at least about 85%, for instance at least about 90%, such as atleast about 95% amino acid sequence identity to a sequence according toany one of SEQ ID Nos: 5, 10, 15, 20 and 25.

In a further embodiment, said variant has at most 1, 2 or 3 amino-acidmodifications, e.g. amino acid substitutions, preferably conservativesubstitutions as compared to said sequence.

In a preferred embodiment, said antibody comprises a human heavy chainvariable region CDR3 sequence comprising an amino acid sequence selectedfrom the group consisting of: SEQ ID NOs: 5, 10, 15, 20 and 25.

In an even further embodiment, the antibody of the invention comprises:

-   -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        3, 4 and 5; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        8, 9 and 10; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        13, 14 and 15; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        18, 19 and 20; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        23, 24 and 25; or    -   a variant of any said VH regions, wherein said variant        preferably only has conservative amino-acid substitutions.

In one embodiment, said variant comprises a VH CDR1 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:3, 8, 13, 18 or 23.

In one embodiment, said variant comprises a VH CDR2 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:4, 9, 14, 19 or 24.

In one embodiment, said variant comprises a VH CDR3 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos: 5, 10, 15, 20 or 25.

In another embodiment, the antibody the antibody comprises

-   -   a) a VL CDR3 region having the sequence as set forth in SEQ ID        NO: 30 and a VH CDR3 region having a sequence selected from the        group consisting of SEQ ID NO: 5,    -   b) a VL CDR3 region having the sequence as set forth in SEQ ID        NO: 35 and a VH CDR3 region having the sequence as set forth in        SEQ ID NO: 10,    -   c) a VL CDR3 region having the sequence as set forth in SEQ ID        NO: 40 and a VH CDR3 region having the sequence as set forth in        SEQ ID NO: 15,    -   d) a VL CDR3 region having the sequence as set forth in SEQ ID        NO: 45 and a VH CDR3 region having the sequence as set forth in        SEQ ID NO: 20,    -   e) a VL CDR3 region having the sequence as set forth in SEQ ID        NO: 50 and a VH CDR3 region having the sequence as set forth in        SEQ ID NO: 25,    -   f) a variant of any of the above, wherein said variant        preferably only has conservative substitutions in said sequences

In one embodiment, said variant comprises a VH CDR3 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos: 5, 10, 15, 20 or 25.In one embodiment, said variant comprises a VL CDR3 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos: 30, 35, 40, 45 or 50;

In a further embodiment, the antibody of the invention comprises:

-   -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        3, 4 and 5, and a VL region comprising the CDR 3 sequence of SEQ        ID NO: 30; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        8, 9 and 10, and a VL region comprising the CDR3 sequence of SEQ        ID NO: 35; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        13, 14 and 15, and and a VL region comprising the CDR3 sequence        of SEQ ID NO: 40; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        18, 19 and 20, and a VL region comprising the CDR3 sequence of        SEQ ID NO: 45; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        23, 24 and 25, and a VL region comprising the CDR3 sequence of        SEQ ID NO: 50; or    -   a variant of any of said antibodies, wherein said variant        preferably only has conservative amino-acid substitutions in        said sequences.

In one embodiment, said variant comprises a VH CDR1 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos: 3, 8, 13, 18 or 23.

In one embodiment, said variant comprises a VH CDR2 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos: 4, 9, 14, 19 or 24.

In one embodiment, said variant comprises a VH CDR3 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:5, 10, 15, 20 or 25.

In one embodiment, said variant comprises a VL CDR1 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:28, 33, 38, 43 or 48.

In one embodiment, said variant comprises a VL CDR2 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:29, 34, 39, 44 or 49.

In one embodiment, said variant comprises a VL CDR3 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:30, 35, 40, 45, or 50.

In a further embodiment, the antibody of the invention comprises:

-   -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        3, 4 and 5 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO: 28, 29 and 30; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        8, 9 and 10 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO: 33, 34 and 35; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        13, 14 and 15 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO: 38, 39 and 40; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        18, 19 and 20 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO: 43, 44 and 45; or    -   a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:        23, 24 and 25 and a VL region comprising the CDR1, 2 and 3        sequences of SEQ ID NO: 48, 49 and 50; or    -   a variant of any of said antibodies, wherein said variant        preferably only has conservative amino acid modifications in        said sequences.

In one embodiment, said variant comprises a VH CDR1 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos: 3, 8, 13, 18 or 23;

In one embodiment, said variant comprises a VH CDR2 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:4, 9, 14, 19 or 24.

In one embodiment, said variant comprises a VH CDR3 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:5, 10, 15, 20 or 25.

In one embodiment, said variant comprises a VL CDR1 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:28, 33, 38, 43 or 48.

In one embodiment, said variant comprises a VL CDR2 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:29, 34, 39, 44 or 49.

In one embodiment, said variant comprises a VL CDR3 which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:30, 35, 40, 45 or 50.

In an even further embodiment, the antibody of the invention comprises:

-   -   a VH region comprising the sequence of SEQ ID NO: 2 and a VL        region comprising the sequence of SEQ ID NO: 27; or    -   a VH region comprising the sequence of SEQ ID NO: 7 and a VL        region comprising the sequence of SEQ ID NO: 32; or    -   a VH region comprising the sequence of SEQ ID NO: 12 and a VL        region comprising the sequence of SEQ ID NO: 37; or    -   a VH region comprising the sequence of SEQ ID NO: 17 and a VL        region comprising the sequence of SEQ ID NO: 42; or    -   a VH region comprising the sequence of SEQ ID NO: 22 and a VL        region comprising the sequence of SEQ ID NO: 47; or    -   a variant of any of the above, wherein said variant preferably        only has conservative modifications.

In one embodiment, said variant comprises a VH region which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID Nos:2, 7, 12, 17 or 22.

In one embodiment, said variant comprises a VL region which consistsessentially of a sequence having at least about 50%, such as at least60%, for instance at least about 70%, such as at least about 75%, forinstance at least about 80%, such as at least about 85%, for instance atleast about 90%, such as at least about 95% amino acid sequence identityto a sequence according to any one of SEQ ID NO: 27, 32, 37, 42 or 47.

In a further embodiment the antibody of the present invention comprisesa VH having at least 80% identity, such as 90%, or 95%, or 97%, or 98%,or 99% identity to a VH region sequence selected from the groupconsisting of: SEQ ID NO: 2, 7, 12, 17 or 22.

In a further embodiment the antibody of the present invention comprisesa VL having at least 80% identity, such as 90%, or 95%, or 97%, or 98%,or 99% identity to a VL region sequence selected from the groupconsisting of: SEQ ID NO: 27, 32, 37, 42 or 47.

In an even further embodiment, the antibody of the invention comprises aVH region selected from the group consisting of: SEQ ID NO: 2, 7, 12, 17or 22.

In an even further embodiment, the antibody of the invention comprises aVL region selected from the group consisting of: SEQ ID NO: 27, 32, 37,42 or 47.

In an even further embodiment, the antibody of the invention comprises:

-   -   a VH region comprising the sequence of SEQ ID NO: 2 and a VL        region comprising the sequence of SEQ ID NO: 27; or    -   a VH region comprising the sequence of SEQ ID NO: 7 and a VL        region comprising the sequence of SEQ ID NO: 32; or    -   a VH region comprising the sequence of SEQ ID NO: 12 and a VL        region comprising the sequence of SEQ ID NO: 37; or    -   a VH region comprising the sequence of SEQ ID NO: 17 and a VL        region comprising the sequence of SEQ ID NO: 42; or    -   a VH region comprising the sequence of SEQ ID NO: 22 and a VL        region comprising the sequence of SEQ ID NO: 47; or

The present invention also, in one aspect, provides anti-CD38 antibodieswhich are characterized with respect to their ability to compete with anantibody having:

-   -   a VH region comprising the sequence of SEQ ID NO: 2 and a VL        region comprising the sequence of SEQ ID NO: 27; or    -   a VH region comprising the sequence of SEQ ID NO: 7 and a VL        region comprising the sequence of SEQ ID NO: 32; or    -   a VH region comprising the sequence of SEQ ID NO: 12 and a VL        region comprising the sequence of SEQ ID NO: 37; or    -   a VH region comprising the sequence of SEQ ID NO: 17 and a VL        region comprising the sequence of SEQ ID NO: 42; or    -   a VH region comprising the sequence of SEQ ID NO: 22 and a VL        region comprising the sequence of SEQ ID NO: 47.

The present invention also relates to provides anti-CD38 antibodieswhich bind to the same epitope as an antibody having:

-   -   a VH region comprising the sequence of SEQ ID NO: 2 and a VL        region comprising the sequence of SEQ ID NO: 27; or    -   a VH region comprising the sequence of SEQ ID NO: 7 and a VL        region comprising the sequence of SEQ ID NO: 32; or    -   a VH region comprising the sequence of SEQ ID NO: 12 and a VL        region comprising the sequence of SEQ ID NO: 37; or    -   a VH region comprising the sequence of SEQ ID NO: 17 and a VL        region comprising the sequence of SEQ ID NO: 42; or    -   a VH region comprising the sequence of SEQ ID NO: 22 and a VL        region comprising the sequence of SEQ ID NO: 47.

The antibody of the invention may be of any isotype. The choice ofisotype typically will be guided by the desired effector functions, suchas ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4.Either of the human light chain constant regions, kappa or lambda, maybe used. If desired, the class of an anti-CD38 antibody of the presentinvention may be switched by known methods. For example, an antibody ofthe present invention that was originally IgM may be class switched toan IgG antibody of the present invention. Further, class switchingtechniques may be used to convert one IgG subclass to another, forinstance from IgG1 to IgG2. Thus, the effector function of theantibodies of the present invention may be changed by isotype switchingto, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody forvarious therapeutic uses. In one embodiment an antibody of the presentinvention is an IgG1 antibody, for instance an IgG1,κ.

In one embodiment, the antibody of the invention is a full-lengthantibody. In another embodiment, the antibody of the invention is anantibody fragment or a single-chain antibody.

Antibody fragments may e.g. be obtained by fragmentation usingconventional techniques, and the fragments screened for utility in thesame manner as described herein for whole antibodies. For example,F(ab′)₂ fragments may be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment may be treated to reduce disulfide bridges toproduce Fab′ fragments. Fab fragments may be obtained by treating an IgGantibody with papain; Fab′ fragments may be obtained with pepsindigestion of IgG antibody. A F(ab′) fragment may also be produced bybinding Fab′ described below via a thioether bond or a disulfide bond. AFab′ fragment is an antibody fragment obtained by cutting a disulfidebond of the hinge region of the F(ab′)₂. A Fab′ fragment may be obtainedby treating a F(ab′)₂ fragment with a reducing agent, such asdithiothreitol. Antibody fragments may also be generated by expressionof nucleic acids encoding such fragments in recombinant cells (see forinstance Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). Forexample, a chimeric gene encoding a portion of a F(ab′)2 fragment couldinclude DNA sequences encoding the CH1 domain and hinge region of the Hchain, followed by a translational stop codon to yield such a truncatedantibody fragment molecule.

In one embodiment, the anti-CD38 antibody is a monovalent antibody,preferably a monovalent antibody as described in WO2007059782 (Genmab)(incorporated herein by reference). Accordingly, in one embodiment, theantibody is a monovalent antibody, wherein said anti-CD38 antibody isconstructed by a method comprising:

i) providing a nucleic acid construct encoding the light chain of saidmonovalent antibody, said construct comprising a nucleotide sequenceencoding the VL region of a selected antigen specific anti-CD38 antibodyand a nucleotide sequence encoding the constant CL region of an Ig,wherein said nucleotide sequence encoding the VL region of a selectedantigen specific antibody and said nucleotide sequence encoding the CLregion of an Ig are operably linked together, and wherein, in case of anIgG1 subtype, the nucleotide sequence encoding the CL region has beenmodified such that the CL region does not contain any amino acidscapable of forming disulfide bonds or covalent bonds with other peptidescomprising an identical amino acid sequence of the CL region in thepresence of polyclonal human IgG or when administered to an animal orhuman being;ii) providing a nucleic acid construct encoding the heavy chain of saidmonovalent antibody, said construct comprising a nucleotide sequenceencoding the VH region of a selected antigen specific antibody and anucleotide sequence encoding a constant CH region of a human Ig, whereinthe nucleotide sequence encoding the CH region has been modified suchthat the region corresponding to the hinge region and, as required bythe Ig subtype, other regions of the CH region, such as the CH3 region,does not comprise any amino acid residues which participate in theformation of disulphide bonds or covalent or stable non-covalentinter-heavy chain bonds with other peptides comprising an identicalamino acid sequence of the CH region of the human Ig in the presence ofpolyclonal human IgG or when administered to an animal human being,wherein said nucleotide sequence encoding the VH region of a selectedantigen specific antibody and said nucleotide sequence encoding the CHregion of said Ig are operably linked together;iii) providing a cell expression system for producing said monovalentantibody;iv) producing said monovalent antibody by co-expressing the nucleic acidconstructs of (i) and (ii) in cells of the cell expression system of(iii).

Similarly, in one embodiment, the anti-CD38 antibody is a monovalentantibody, which comprises

(i) a variable region of an antibody of the invention as describedherein or an antigen binding part of the said region, and(ii) a C_(H) region of an immunoglobulin or a fragment thereofcomprising the C_(H)2 and C_(H)3 regions, wherein the C_(H) region orfragment thereof has been modified such that the region corresponding tothe hinge region and, if the immunoglobulin is not an IgG4 subtype,other regions of the C_(H) region, such as the C_(H)3 region, do notcomprise any amino acid residues, which are capable of forming disulfidebonds with an identical C_(H) region or other covalent or stablenon-covalent inter-heavy chain bonds with an identical C_(H) region inthe presence of polyclonal human IgG.

In a further embodiment, the heavy chain of the monovalent anti-CD38antibody has been modified such that the entire hinge has been deleted.

In a further embodiment, said monovalent antibody is of the IgG4subtype, but the C_(H)3 region has been modified so that one or more ofthe following amino acid substitutions have been made: Thr (T) inposition 366 has been replaced by Ala (A); Leu (L) in position 368 hasbeen replaced by Ala (A); Leu (L) in position 368 has been replaced byVal (V); Phe (F) in position 405 has been replaced by Ala (A); Phe (F)in position 405 has been replaced by Leu (L); Tyr (Y) in position 407has been replaced by Ala (A); Arg (R) in position 409 has been replacedby Ala (A).

In another further embodiment, the sequence of said monovalent antibodyhas been modified so that it does not comprise any acceptor sites forN-linked glycosylation.

Anti-CD38 antibodies of the invention also include single chainantibodies. Single chain antibodies are peptides in which the heavy andlight chain Fv regions are connected. In one embodiment, the presentinvention provides a single-chain Fv (scFv) wherein the heavy and lightchains in the Fv of an anti-CD38 antibody of the present invention arejoined with a flexible peptide linker (typically of about 10, 12, 15 ormore amino acid residues) in a single peptide chain. Methods ofproducing such antibodies are described in for instance U.S. Pat. No.4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994), Bird et al., Science 242, 423-426 (1988), Huston et al., PNASUSA 85, 5879-5883 (1988) and McCafferty et al., Nature 348, 552-554(1990). The single chain antibody may be monovalent, if only a singleV_(H) and V_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used.

In one embodiment, the anti-CD38 antibody of the invention is aneffector-function-deficient antibody. Such antibodies are particularlyuseful when the antibody is for use in stimulation and demping of theimmune system through blocking of the inhibitory effects of CD38. Forsuch applications, it may be advantageous that the antibody has noeffector functions, such as ADCC, as this may lead to undesiredcytotoxicity.

In one embodiment, the effector-function-deficient anti-CD38 antibody isa stabilized IgG4 antibody. Examples of suitable stabilized IgG4antibodies are antibodies, wherein arginine at position 409 in a heavychain constant region of human IgG4, which is indicated in the EU indexas in Kabat et al., is substituted with lysine, threonine, methionine,or leucine, preferably lysine (described in WO2006033386 (Kirin)).Preferably, said antibody comprises a Lys or Ala residue at the positioncorresponding to 409 or the CH3 region of the antibody has been replacedby the CH3 region of human IgG1, of human IgG2 or of human IgG3.

In a further embodiment. the stabilized IgG4 anti-CD38 antibody is anIgG4 antibody comprising a heavy chain and a light chain, wherein saidheavy chain comprises a human IgG4 constant region having a residueselected from the group consisting of: Lys, Ala, Thr, Met and Leu at theposition corresponding to 409 and/or a residue selected from the groupconsisting of: Ala, Val, Gly, Ile and Leu at the position correspondingto 405, and wherein said antibody optionally comprises one or morefurther substitutions, deletions and/or insertions, but does notcomprise a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably,said antibody comprises a Lys or Ala residue at the positioncorresponding to 409 or the CH3 region of the antibody has been replacedby the CH3 region of human IgG1, of human IgG2 or of human IgG3.

In an even further embodiment. the stabilized IgG4 anti-CD38 antibody isan IgG4 antibody comprising a heavy chain and a light chain, whereinsaid heavy chain comprises a human IgG4 constant region having a residueselected from the group consisting of: Lys, Ala, Thr, Met and Leu at theposition corresponding to 409 and/or a residue selected from the groupconsisting of: Ala, Val, Gly, Ile and Leu at the position correspondingto 405, and wherein said antibody optionally comprises one or morefurther substitutions, deletions and/or insertions and wherein saidantibody comprises a Cys-Pro-Pro-Cys sequence in the hinge region.Preferably, said antibody comprises a Lys or Ala residue at the positioncorresponding to 409 or the CH3 region of the antibody has been replacedby the CH3 region of human IgG1, of human IgG2 or of human IgG3.

In a further embodiment, the effector-function-deficient anti-CD38antibody is an antibody of a non-IgG4 type, e.g. IgG1, IgG2 or IgG3which has been mutated such that the ability to mediate effectorfunctions, such as ADCC, has been reduced or even eliminated. Examplesof such mutations have e.g. been described in Dall'Acqua W F et al., JImmunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.;75(24):12161-12168 (2001).

Conjugates

In a further embodiment. the antibody of the invention is conjugated toanother moiety, such as a cytotoxic moiety, a radioisotope or a drug.

Such antibodies may be produced by chemically conjugating the othermoiety to the N-terminal side or C-terminal side of the anti-CD38antibody or fragment thereof (e.g., an anti-CD38 antibody H chain, Lchain, or anti-CD38 specific/selective fragment thereof) (see, e.g.,Antibody Engineering Handbook, edited by Osamu Kanemitsu, published byChijin Shokan (1994)). Such conjugated antibody derivatives may also begenerated by conjugation at internal residues or sugars, whereappropriate.

Anti-CD38 antibodies described herein may also be modified by inclusionof any suitable number of modified amino acids. Suitability in thiscontext is generally determined by the ability to at least substantiallyretain CD38 selectivity and/or specificity associated with thenon-derivatized parent anti-CD38 antibody. The inclusion of one or moremodified amino acids may be advantageous in, for example, increasingpolypeptide serum half-life, reducing polypeptide antigenicity, orincreasing polypeptide storage stability. Amino acid(s) are modified,for example, co-translationally or post-translationally duringrecombinant production (e.g., N-linked glycosylation at N-X-S/T motifsduring expression in mammalian cells) or modified by synthetic means.Non-limiting examples of a modified amino acid include a glycosylatedamino acid, a sulfated amino acid, a prenylated (e.g., farnesylated,geranylgeranylated) amino acid, an acetylated amino acid, an acylatedamino acid, a PEGylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like.References adequate to guide one of skill in the modification of aminoacids are replete throughout the literature. Example protocols are foundin Walker (1998) Protein Protocols On Cd-Rom, Humana Press, Towata, N.J.

Anti-CD38 antibodies may also be chemically modified by covalentconjugation to a polymer to for instance increase their circulatinghalf-life. Exemplary polymers, and methods to attach them to peptides,are illustrated in for instance U.S. Pat. Nos. 4,766,106, 4,179,337,4,495,285 and 4,609,546.

In one embodiment, the present invention provides an anti-CD38 antibodythat is conjugated to a second molecule that is selected from aradionuclide, an enzyme, an enzyme substrate, a cofactor, a fluorescentmarker, a chemiluminescent marker, a peptide tag, or a magneticparticle. In one embodiment, an anti-CD38 antibody may be conjugated toone or more antibody fragments, nucleic acids (oligonucleotides),nucleases, hormones, immunomodulators, chelators, boron compounds,photoactive agents, dyes, and the like. These and other suitable agentsmay be coupled either directly or indirectly to an anti-CD38 antibody ofthe present invention. One example of indirect coupling of a secondagent is coupling by a spacer moiety.

In one embodiment, anti-CD38 antibodies comprising one or moreradiolabeled amino acids are provided. A radiolabeled anti-CD38 antibodymay be used for both diagnostic and therapeutic purposes (conjugation toradiolabeled molecules is another possible feature). Nonlimitingexamples of labels for polypeptides include, but are not limited to 3H,14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re. Methods forpreparing radiolabeled amino acids and related peptide derivatives areknown in the art (see for instance Junghans et al., in CancerChemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo,eds., Lippincott Raven (1996)) and U.S. Pat. Nos. 4,681,581, 4,735,210,5,101,827, 5,102,990 (U.S. RE35,500), 5,648,471 and 5,697,902.

In one embodiment, the anti-CD38 antibody of the invention is conjugatedto a radioisotope or to a radioisotope-containing chelate. For example,the anti-CD38 antibody can be conjugated to a chelator linker, e.g.DOTA, DTPA or tiuxetan, which allows for the anti-CD38 antibody to becomplexed with a radioisotope. The anti-CD38 antibody may also oralternatively comprise or be conjugated to one or more radiolabeledamino acids or other radiolabeled molecule. A radiolabeled anti-CD38antibody may be used for both diagnostic and therapeutic purposes.Non-limiting examples of radioisotopes include 3H, 14C, 15N, 35S, 90Y,99Tc, 125I, 111In, 131I, 186Re, 213Bs, 225Ac and 227Th.

In one embodiment, the anti-CD38 antibody of the invention is conjugatedto auristatins or auristatin peptide analogs and derivates (U.S. Pat.Nos. 5,635,483; 5,780,588). Auristatins have been shown to interferewith microtubule dynamics, GTP hydrolysis and nuclear and cellulardivision (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and antifungalactivity (Pettit et al., (1998) Antimicrob. Agents and Chemother.42:2961-2965. The auristatin drug moiety may be attached to theantibody, via an linker, through the N (amino) terminus or the C(terminus) of the peptidic drug moiety.

Exemplary auristatin embodiments include the N-terminus-linkedmonomethyl auristatin drug moieties DE and DF, disclosed in Senter etal., Proceedings of the American Association for Cancer Research. Volume45, abstract number 623, presented Mar. 28, 2004 and described in US2005/0238648).

An exemplary auristatin embodiment is MMAE (monomethyl auristatin E),wherein the wavy line indicates the covalent attachment to the linker(L) of an antibody drug conjugate:

Another exemplary auristatin embodiment is MMAF (monomethyl auristatinF), wherein the wavy line indicates the covalent attachment to a linker(L) of an antibody drug conjugate (US2005/0238649):

The anti-CD38 antibody drug conjugates according to the inventioncomprise a linker unit between the cytostatic drug unit and the antibodyunit. In some embodiments, the linker is cleavable under intracellularconditions, such that the cleavage of the linker releases the drug unitfrom the antibody in the intracellular environment. In yet anotherembodiment, the linker unit is not cleavable and the drug is forinstance released by antibody degradation. In some embodiments, thelinker is cleavable by a cleavable agent that is present in theintracellular environment (e.g. within a lysosome or endosome orcaveola). The linker can be, e.g. a peptidyl linker that is cleaved byan intracellular peptidase or protease enzyme, including but not limitedto, a lysosomal or endosomal protease. In some embodiments, the peptidyllinker is at least two amino acids long or at least three amino acidslong. Cleaving agents can include cathepsins B and D and plasmin, all ofwhich are known to hydrolyze dipeptide drug derivatives resulting in therelease of active drug inside the target cells (see e.g. Dubowchik andWalker, 1999, Pharm. Therapeutics 83:67-123). In a specific embodiment,the peptidyl linker cleavable by an intracellular protease is a Val-Cit(valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker(see e.g. U.S. Pat. No. 6,214,345, which describes the synthesis ofdoxorubicin with the Val-Cit linker). An advantage of usingintracellular proteolytic release of the therapeutic agent is that theagent is typically attenuated when conjugated and the serum stabilitiesof the conjugates are typically high.

In yet another embodiment, the linker unit is not cleavable and the drugis released by antibody degradation (see US 2005/0238649). Typically,such a linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment” in the context of a linker means that no morethan 20%, typically no more than about 15%, more typically no more thanabout 10%, and even more typically no more than about 5%, no more thanabout 3%, or no more than about 1% of the linkers, in a sample ofantibody drug conjugate compound, are cleaved when the antibody drugconjugate compound presents in an extracellular environment (e.g.plasma). Whether a linker is not substantially sensitive to theextracellular environment can be determined for example by incubatingwith plasma the antibody drug conjugate compound for a predeterminedtime period (e.g. 2, 4, 8, 16 or 24 hours) and then quantitating theamount of free drug present in the plasma.

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components have the following structures (wherein Ab meansantibody and p, representing the drug-loading (or average number ofcytostatic drugs per molecule), is 1 to about 8).

Examples where a cleavable linker is combined with an auristatin includevcMMAF and vcMMAE (vc is the abbreviation for the Val-Cit(valine-citruline) based linker):

Other examples include auristatins combined with a non-cleavable linker,such as mcMMAF. (mc is an abbreviation of maleimido caproyl):

The cytostatic drug loading is represented by p and is the averagenumber of cytostatic drug moieties per antibody in a molecule (alsodesignated as the drug to antibody ratio, DAR). The cytostatic drugloading may range from 1 to 20 drug moieties per antibody and may occuron amino acids with useful functional groups such as, but not limitedto, amino or sulfhydryl groups, as in lysine or cysteine.

Depending on the way of conjugation, p may be limited by the number ofattachment sites on the antibody, for example where the attachment is acysteine thiol, as in the present invention. Generally, antibodies donot contain many free and reactive cysteine thiol groups which may belinked to a drug moiety as most cysteine thiol residues in antibodiesexist as disulfide bridges. Therefore, in certain embodiments, anantibody may be reduced with reducing agent such as dithiothreitol (DTT)or tricarbonylethylphosphine (TCEP), under partial or fully reducingconditions, to generate reactive cysteine thiol groups. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8, as a maximum of 8 free cysteine thiol groups becomesavailable after (partial) reduction of the antibody (there are 8cysteines involved in inter-chain disulfide bonding).

In one embodiment, the drug linker moiety is vcMMAE. The vcMMAE druglinker moiety and conjugation methods are disclosed in WO2004010957,U.S. Pat. Nos. 7,659,241, 7,829,531, 7,851,437 and U.S. Ser. No.11/833,028 (Seattle Genetics, Inc.), (which are incorporated herein byreference), and the vcMMAE drug linker moiety is bound to the anti-CD38antibodies at the cysteines using a method similar to those disclosed intherein.

In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF druglinker moiety and conjugation methods are disclosed in U.S. Pat. No.7,498,298, U.S. Ser. No. 11/833,954, and WO2005081711 (Seattle Genetics,Inc.) (which are incorporated herein by reference), and the mcMMAF druglinker moiety is bound to the anti-CD38 antibodies at the cysteinesusing a method similar to those disclosed in therein.

Upon purifying the anti-CD38 antibody drug conjugates they may beformulated into pharmaceutical compositions using well knownpharmaceutical carriers or excipients.

In one embodiment, an anti-CD38 antibody is conjugated to a functionalnucleic acid molecule. Functional nucleic acids include antisensemolecules, interfering nucleic acid molecules (e.g., siRNA molecules),aptamers, ribozymes, triplex forming molecules, and external guidesequences. External guide sequences (EGSs) are molecules that bind atarget nucleic acid molecule forming a complex that is recognized byRNase P, which cleaves the target molecule. The functional nucleic acidmolecules may act as effectors, inhibitors, modulators, and stimulatorsof a specific activity possessed by a target molecule, or the functionalnucleic acid molecules may possess a de novo activity independent of anyother molecules. A representative sample of methods and techniques whichaid in the design and use of antisense molecules may be found in thefollowing non-limiting list of US patents: U.S. Pat. Nos. 5,135,917,5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138,5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, 5,994,320,5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042,6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319 and 6,057,437.

Any method known in the art for conjugating the anti-CD38 antibody tothe conjugated molecule(s), such as those described above, may beemployed, including those methods described by Hunter et al., Nature144, 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain etal., J. Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. andCytochem. 30, 407 (1982). Linkage/conjugation may be accomplished in anysuitable way. For example, a covalent linkage may take the form of adisulfide bond (if necessary and suitable, an anti-CD38 antibody couldbe engineered to contain an extra cysteine codon. A toxin molecule,derivatized with a sulfhydryl group reactive with the cysteine of themodified anti-CD38 antibody, may form an immunoconjugate with theanti-CD38 antibody. Alternatively, a sulfhydryl group may be introduceddirectly to an anti-CD38 antibody using solid phase polypeptidetechniques. For example, the introduction of sulfhydryl groups intopeptides is described by Hiskey, Peptides 3, 137 (1981). Theintroduction of sulfhydryl groups into proteins is described in Maasenet al., Eur. J. Biochem. 134, 32 (1983).

Numerous types of cytotoxic compounds may be joined to proteins throughthe use of a reactive group on the cytotoxic compound or through the useof a cross-linking agent. A common reactive group that will form astable covalent bond in vivo with an amine is isothiocyanate (Means etal., Chemical modifications of proteins (Holden-Day, San Francisco 1971)pp. 105-110). This group preferentially reacts with the ε-amine group oflysine. Maleimide is a commonly used reactive group to form a stable invivo covalent bond with the sulfhydryl group on cysteine (Ji., MethodsEnzymol 91, 580-609 (1983)). Monoclonal antibodies typically areincapable of forming covalent bonds with radiometal ions, but they maybe attached to the antibody indirectly through the use of chelatingagents that are covalently linked to the antibodies. Chelating agentsmay be attached through amines (Meares et al., Anal. Biochem. 142, 68-78(1984)) and sulfhydral groups (Koyama, Chem. Abstr. 120, 217262t (1994))of amino acid residues and also through carbohydrate groups (Rodwell etal., PNAS USA 83, 2632-2636 (1986), Quadri et al., Nucl. Med. Biol. 20,559-570 (1993)). A therapeutic or diagnostic agent may also oralternatively be attached at the hinge region of a reduced antibodycomponent via disulfide bond formation.

In one embodiment, the present invention provides an anti-CD38 antibody,such as a human anti-CD38 antibody, conjugated to a therapeutic moiety,such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or aradioisotope. Such conjugates are referred to herein as“immunoconjugates”. Immunoconjugates which include one or morecytotoxins are referred to as “immunotoxins”. A cytotoxin or cytotoxicagent includes any agent that is detrimental to (e.g., kills) cells. Fora description of these classes of drugs which are well known in the art,and their mechanisms of action, see Goodman et al., Goodman and Gilman'sThe Pharmacological Basis Of Therapeutics, 8th Ed., Macmillan PublishingCo., 1990. Additional techniques relevant to the preparation of antibodyimmunotoxins are provided in for instance Vitetta, Immunol. Today 14,252 (1993) and U.S. Pat. No. 5,194,594.

Suitable therapeutic agents for forming immunoconjugates of the presentinvention include taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydro-testosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin, antimetabolites (such as methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine,hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents(such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatinand other platinum derivatives, such as carboplatin), antibiotics (suchas dactinomycin (formerly actinomycin), bleomycin, daunorubicin(formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin,mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin andrelated molecules (such as diphtheria A chain and active fragmentsthereof and hybrid molecules), ricin toxin (such as ricin A or adeglycosylated ricin A chain toxin), cholera toxin, a Shiga-like toxin(SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussistoxin, tetanus toxin, soybean Bowman-Birk protease inhibitor,Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S),Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycintoxins, calicheamicins and duocarmycins. Therapeutic agents, which maybe administered in combination with a an anti-CD38 antibody of thepresent invention as described elsewhere herein, may also be candidatesfor therapeutic moieties useful for conjugation to an anti-CD38 antibodyof the present invention.

As indicated above, the drug moiety need not be construed as limited toclassical chemical therapeutic agents. For example, the drug moiety maybe a protein or polypeptide possessing a desired biological activity. Inone embodiment, the anti-CD38 antibody of the present invention isattached to a chelator linker, e.g. tiuxetan, which allows for theantibody to be conjugated to a radioisotope.

Bispecific Antibodies

In a further aspect, the invention relates to a bispecific moleculecomprising a first antigen binding site from an anti-CD38 antibody ofthe invention as described herein above and a second antigen bindingsite with a different binding specificity, such as a binding specificityfor a human effector cell, a human Fc receptor, a T cell receptor, a Bcell receptor or a binding specificity for a non-overlapping epitope ofCD38, i.e. a bispecific antibody wherein the first and second antigenbinding sites do not cross-block each other for binding to CD38, e.g.when tested as described in Example 3.

Exemplary bispecific antibody molecules of the invention comprise (i)two antibodies, one with a specificity to CD38 and another to a secondtarget that are conjugated together, (ii) a single antibody that has onechain or arm specific to CD38 and a second chain or arm specific to asecond molecule, (iii) a single chain antibody that has specificity toCD38 and a second molecule, e.g., via two scFvs linked in tandem by anextra peptide linker; (iv) a dual-variable-domain antibody (DVD-Ig),where each light chain and heavy chain contains two variable domains intandem through a short peptide linkage (Wu et al., Generation andCharacterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™)Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010));(v) a chemically-linked bispecific (Fab′)₂ fragment; (vi) a Tandab,which is a fusion of two single chain diabodies resulting in atetravalent bispecific antibody that has two binding sites for each ofthe target antigens; (vii) a flexibody, which is a combination of scFvswith a diabody resulting in a multivalent molecule; (viii) a so called“dock and lock” molecule, based on the “dimerization and docking domain”in Protein Kinase A, which, when applied to Fabs, can yield a trivalentbispecific binding protein consisting of two identical Fab fragmentslinked to a different Fab fragment; (ix) a so-called Scorpion molecule,comprising, e.g., two scFvs fused to both termini of a human Fc-region;and (x) a diabody. In one embodiment, the bispecific antibody of thepresent invention is a diabody, a cross-body, or a bispecific obtainedvia a controlled Fab arm exchange as those described in the presentinvention.

Examples of platforms useful for preparing bispecific antibodies includebut are not limited to BITE (Micromet), DART (MacroGenics), Fcab andMab² (F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab armexchange, Genmab, this application).

Examples of different classes of bispecific antibodies include but arenot limited to

-   -   asymmetric IgG-like molecules, wherein the one side of the        molecule contains the Fab region or part of the Fab region of at        least one antibody, and the other side of the molecule contains        the Fab region or parts of the Fab region of at least one other        antibody; in this class, asymmetry in the Fc region could also        be present, and be used for specific linkage of the two parts of        the molecule;    -   symmetric IgG-like molecules, wherein the two sides of the        molecule each contain the Fab region or part of the Fab region        of at least two different antibodies;    -   IgG fusion molecules, wherein full length IgG antibodies are        fused to extra Fab regions or parts of Fab regions;    -   Fc fusion molecules, wherein single chain Fv molecules or        stabilized diabodies are fused to Fcγ regions or parts thereof;    -   Fab fusion molecules, wherein different Fab-fragments are fused        together;    -   ScFv- and diabody-based molecules wherein different single chain        Fv molecules or different diabodies are fused to each other or        to another protein or carrier molecule.

Examples of asymmetric IgG-like molecules include but are not limited tothe Triomab/Quadroma (Trion Pharma/Fresenius Biotech), theKnobs-into-Holes (Genentech), CrossMAbs (Roche) and theelectrostatically-matched (Amgen), the LUZ-Y (Genentech), the StrandExchange Engineered Domain body (EMD Serono), the Biclonic (Merus) andthe DuoBody (Genmab A/S).

Example of symmetric IgG-like molecules include but are not limited toDual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech),Cross-linked Mabs (Karmanos Cancer Center), mAb² (F-Star) and CovX-body(CovX/Pfizer).

Examples of IgG fusion molecules include but are not limited to DualVariable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (ImClone/EliLilly), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (BiogenIdec) and TvAb (Roche).

Examples of Fc fusion molecules include but are not limited to ScFv/FcFusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion,Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART)(MacroGenics) and Dual(ScFv)₂-Fab (National Research Center for AntibodyMedicine—China).

Examples of class V bispecific antibodies include but are not limited toF(ab)₂ (Medarex/Amgen), Dual-Action or Bis-Fab (Genentech),Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) andFab-Fv (UCB-Celltech).

Examples of ScFv- and diabody-based molecules include but are notlimited to Bispecific T Cell Engager (BITE) (Micromet9, Tandem Diabody(Tandab) (Affimed), Dual Affinity Retargeting Technology (DART)(MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies(AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) andCOMBODY (Epigen Biotech).

In a further aspect, the invention relates to a bispecific moleculecomprising an anti-CD38 antibody of the invention as described hereinabove and a second binding specificity such as a binding specificity forhuman cytokines. In one embodiment, said cytokine is ananti-inflammatory cytokine such as IL-1ra, IL-4, IL-6, IL-10, IL-11,IL-13, IL-16, IFN-alpha and TGF-beta. In another embodiment saidcytokine is a pro-inflammatory cytokine such as IL-1alpha, IL-1beta andIL-6. In an embodiment the binding specificity is for a human effectorcell, a human Fc receptor or a T cell receptor. In one embodiment, saidT cell receptor is CD3. In another embodiment, said human Fc receptor ishuman FcγRI (CD64), human FcγRII (CD32), FcγRIII (CD16) or a human Fcαreceptor (CD89). Bispecific molecules of the present invention mayfurther include a third binding specificity, in addition to an anti-CD38binding specificity and a binding specificity for a human effector cell,a human Fc receptor or a T cell receptor.

Exemplary bispecific antibody molecules of the invention comprise (i)two antibodies one with a specificity to CD38 and another to a secondtarget that are conjugated together, (ii) a single antibody that has onechain specific to CD38 and a second chain specific to a second molecule,and (iii) a single chain antibody that has specificity to CD38 and asecond molecule. In one embodiment, the second molecule is a cancerantigen/tumor-associated antigen such as CD20, carcinoembryonic antigen(CEA), prostate specific antigen (PSA), RAGE (renal antigen),α-fetoprotein, CAMEL (CTL-recognized antigen on melanoma), CT antigens(such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2,GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125,etc.), ganglioside antigens, tyrosinase, gp75, C-myc, Marti, MelanA,MUM-1, MUM-2, MUM-3, HLA-B7, and Ep-CAM. In one embodiment, the secondmolecule is a cancer-associated integrin, such as α5β3 integrin. In oneembodiment, the second molecule is an angiogenic factor or othercancer-associated growth factor, such as a vascular endothelial growthfactor (VEGF), a fibroblast growth factor (FGF), epidermal growth factor(EGF), epidermal growth factor receptor (EGFR), angiogenin, andreceptors thereof, particularly receptors associated with cancerprogression (for instance one of the HER1-HER4 receptors). Other cancerprogression-associated proteins discussed herein may also be suitablesecond molecules.

In an embodiment of the invention, the antibody is a single antibodythat has one chain specific to the CD38 epitope described in thisinvention comprising aspartic acid at position 202 and a second chainspecific for a CD38 specific epitope that does not comprise the asparticacid at position 202 (a non-competing antibody). Such antibody isdescribed for example in WO2006099875 as antibody 003.

In one embodiment, a bispecific antibody of the present invention is adiabody.

Generation of Bispecific Antibodies by 2-MEA-Induced Fab-Arm Exchange

An in vitro method for producing bispecific antibodies is described inWO 2008119353 (Genmab) and reported by van der Neut-Kolfschoten et al.(Science. 2007 Sep. 14; 317(5844):1554-7). Herein, a bispecific antibodyis formed by “Fab-arm” or “half-molecule” exchange (swapping of a heavychain and attached light chain) between two monospecific IgG4- orIgG4-like antibodies upon incubation under mildly reducing conditions.This Fab-arm exchange reaction is the result of a disulfide-bondisomerization reaction wherein the inter heavy-chain disulfide bonds inthe hinge regions of monospecific antibodies are reduced and theresulting free cysteines form a new inter heavy-chain disulfide bondwith cysteine residues of another antibody molecule with a differentspecificity. The resulting product is a bispecific antibody having twoFab arms with different sequences.

In a novel invention the knowledge of this natural IgG4 Fab-arm exchangeis adapted to generate a method to produce stable IgG1-based bispecificantibodies. The bispecific antibody product generated by this methoddescribed below will no longer participate in IgG4 Fab-arm exchange. Thebasis for this method is the use of complimentary CH3 domains, whichpromote the formation of heterodimers under specific assay conditions.To enable the production of bispecific antibodies by this method, IgG1molecules carrying certain mutations in the CH3 domain were generated:in one of the parental IgG1 antibody T350I, K370T and F405L mutations inthe other parental IgG1 antibody the K409R mutation.

To generate bispecific antibodies, these two parental antibodies, eachantibody at a final concentration of 0.5 mg/mL (equimolarconcentration), were incubated with 25 mM 2-mercaptoethylamine-HCl(2-MEA) in a total volume of 100 μL TE at 37° C. for 90 min. Thereduction reaction is stopped when the reducing agent 2-MEA is removedby using spin columns (Microcon centrifugal filters, 30 k, Millipore)according to the manufacturer's protocol. By this method the followingbispecific antibodies may be generated:

A bispecfic antibody wherein the anti-CD38 antibody is 025, 026, 028,049 or 056, and the second binding moiety is an anti-CD3 antibody.

A bispecfic antibody wherein the anti-CD38 antibody is 025, 026, 028,049 or 056, and the second binding moiety is an anti-CD20 antibody, suchas ofatumumab.

A bispecfic antibody wherein the anti-CD38 antibody is 025, 026, 028,049 or 056, and the second binding moiety is an anti-CD16 antibody.

A bispecfic antibody wherein the anti-CD38 antibody is 025, 026, 028,049 or 056, and the second binding moiety is an anti-CD32 antibody.

A bispecfic antibody wherein the anti-CD38 antibody is 025, 026, 028,049 or 056, and the second binding moiety is an anti-CD64 antibody.

Nucleic Acids, Vectors, Host Cells and Method for Producing Antibodiesof the Invention

In a further aspect, the invention relates to nucleic acids encoding(parts of) an antibody of the invention and to expression vectorscomprising such nucleic acids.

In one embodiment, the expression vector of the invention comprises anucleotide sequence encoding one or more of the amino acid sequencesselected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 5.

In a further embodiment, the expression vector further comprises anucleotide sequence encoding the constant region of a light chain, aheavy chain or both light and heavy chains of an antibody, e.g. a humanantibody.

Such expression vectors may be used for recombinant production ofantibodies of the invention.

An expression vector in the context of the present invention may be anysuitable vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ananti-CD38 antibody-encoding nucleic acid is comprised in a naked DNA orRNA vector, including, for example, a linear expression element (asdescribed in for instance Sykes and Johnston, Nat Biotech 17, 355-59(1997)), a compacted nucleic acid vector (as described in for instanceU.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such aspBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleicacid vector (as described in for instance Schakowski et al., Mol Ther 3,793-800 (2001)), or as a precipitated nucleic acid vector construct,such as a CaP04-precipitated construct (as described in for instance WO00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler etal., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics7, 603 (1981)). Such nucleic acid vectors and the usage thereof are wellknown in the art (see for instance U.S. Pat. Nos. 5,589,466 and5,973,972).

In one embodiment, the vector is suitable for expression of theanti-CD38 antibody in a bacterial cell. In another embodiment, theexpression vector may be a vector suitable for expression in a yeastsystem. Most typically, the vector will be a vector suitable forexpression of the antibody of the invention in a mammalian cell, such asa CHO, HEK or PER.C6® cell (human cell line developed by DSM and CrucellN.V., the Netherlands). Another suitable vector system is the glutaminesynthetase (GS) vector system developed by Lonza Biologics (see e.g.EP216846, U.S. Pat. No. 5,981,216, WO8704462, EP323997, U.S. Pat. Nos.5,591,639, 5,658,759, EP338841, U.S. Pat. Nos. 5,879,936, and5,891,693).

In an expression vector of the invention, anti-CD38 antibody-encodingnucleic acids may comprise or be associated with any suitable promoter,enhancer, and other expression-facilitating elements. Examples of suchelements include strong expression promoters (e.g., human CMV IEpromoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTRpromoters), effective poly (A) termination sequences, an origin ofreplication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE.

In one embodiment, the anti-CD38-antibody-encoding expression vector maybe positioned in and/or delivered to the host cell or host animal via aviral vector.

In an even further aspect, the invention relates to a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces an antibody of the invention as defined herein. Examples ofhost cells include yeast, bacterial, and mammalian cells, such as a CHO,HEK or PER.C6® cells. For example, in one embodiment, the presentinvention provides a cell comprising a nucleic acid stably integratedinto the cellular genome that comprises a sequence coding for expressionof an anti-CD38 antibody of the present invention. In anotherembodiment, the present invention provides a cell comprising anon-integrated nucleic acid, such as a plasmid, cosmid, phagemid, orlinear expression element, which comprises a sequence coding forexpression of an anti-CD38 antibody of the invention.

In a further aspect, the invention relates to a hybridoma which producesan antibody of the invention as defined herein. In an even furtheraspect, the invention relates to a transgenic non-human animalcomprising nucleic acids encoding a human heavy chain and a human lightchain, wherein the animal or plant produces an antibody of theinvention. Generation of such hybridomas and transgenic animals has beendescribed above.

In a further aspect, the invention relates to a method for producing ananti-CD38 antibody of the invention, said method comprising the stepsof:

a) culturing a hybridoma or a host cell of the invention as describedherein above, andb) purifying the antibody of the invention from the culture media.

Pharmaceutical Compositions

In an even further aspect, the invention relates to a pharmaceuticalcomposition comprising:

-   -   an anti-CD38 antibody as defined herein, and    -   a pharmaceutically-acceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 19thEdition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. Apharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e.g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the chosencompound of the present invention and the chosen mode of administration.Suitability for carriers and other components of pharmaceuticalcompositions is determined based on the lack of significant negativeimpact on the desired biological properties of the chosen compound orpharmaceutical composition of the present invention (e.g., less than asubstantial impact (10% or less relative inhibition, 5% or less relativeinhibition, etc.)) on antigen binding.

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the amide thereof, the route of administration,the time of administration, the rate of excretion of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompositions employed, the age, sex, weight, condition, general healthand prior medical history of the patient being treated, and like factorswell known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering a compound of the presentinvention in vivo and in vitro are well known in the art and may beselected by those of ordinary skill in the art.

In one embodiment, a pharmaceutical composition of the present inventionis administered parenterally.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and include epidermal,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment that pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

In one embodiment the compounds of the present invention areadministered in crystalline form by subcutaneous injection, cf. Yang etal., PNAS USA 100(12), 6934-6939 (2003).

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a compound of thepresent invention.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thecompounds of the present invention may be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Methods for the preparation of suchformulations are generally known to those skilled in the art. See e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978.

The pharmaceutical composition of the present invention may contain oneantibody of the present invention or a combination of two or moreantibodies of the present invention.

Therapeutic Uses

In another aspect, the invention relates to the antibody of theinvention as defined herein for use as a medicament.

The anti-CD38 antibodies of the present invention have numeroustherapeutic utilities involving the treatment of disorders involvingcells expressing CD38. For example, the antibodies may be administeredto cells in culture, e.g., in vitro or ex vivo, or to human subjects,e.g., in vivo, to treat or prevent a variety of disorders. As usedherein, the term “subject” is intended to include human and non-humananimals which respond to the antibody. Subjects may for instance includehuman patients having disorders that may be corrected or ameliorated bymodulating CD38 function, such as enzymatic activity, signaltransduction, induction of cytokine expression, induction ofproliferation or differentiation, and/or induction of lysis and/oreliminating/reducing the number of CD38 expressing cells.

For example, the anti-CD38 antibodies may be used to elicit in vivo orin vitro one or more of the following biological activities: modulatingCD38 function (such as enzymatic activity, signal transduction,induction of cytokine expression, induction of proliferation ordifferentiation, and/or induction of lysis), killing a cell expressingCD38, mediating phagocytosis or ADCC of a cell expressing CD38 in thepresence of human effector cells, and by mediating CDC of a cellexpressing CD38 in the presence of complement or by killing CD38expressing cells by apoptosis.

The present invention provides methods for treating or preventing adisorder involving cells expressing CD38 in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-CD38 antibody of the present invention to a subject in needthereof. Such a method involves administering to a subject an anti-CD38antibody of the present invention in an amount effective to treat orprevent the disorder.

In one embodiment of the present invention, the disorder involving cellsexpressing CD38 may be cancer, i.e. a tumorigenic disorder, such as adisorder characterized by the presence of tumor cells expressing CD38including, for example, B cell lymphoma, plasma cell malignancies, T/NKcell lymphoma and myeloid malignancies.

Examples of such tumorigenic diseases include B cell lymphomas/leukemiasincluding precursor B cell lymphoblastic leukemia/lymphoma and B cellnon-Hodgkin's lymphomas; acute promyelocytic leukemia, acutelymphoblastic leukemia and mature B cell neoplasms, such as B cellchronic lymhocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), Bcell acute lymphocytic leukemia, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicularlymphoma (FL), including low-grade, intermediate-grade and high-gradeFL, cutaneous follicle center lymphoma, marginal zone B cell lymphoma(MALT type, nodal and splenic type), hairy cell leukemia, diffuse largeB cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma,plasma cell leukemia, post-transplant lymphoproliferative disorder,Waldenström's macroglobulinemia, plasma cell leukemias and anaplasticlarge-cell lymphoma (ALCL).

In one embodiment, the disorder involving cells expressing CD38 ismultiple myeloma.

In one embodiment, the disorder involving cells expressing CD38 isselected from chronic lymphocytic leukemia (CLL), acute lymphoblasticleukemia (ALL), acute myelogenous leukemia (adults) (AML), mantle celllymphoma, follicular lymphoma, and diffuse large B-cell lymphoma.

In one embodiment the disorder involving cells expressing CD38 isnon-small cell lung cancer (NSCLC).

Examples of B cell non-Hodgkin's lymphomas are lymphomatoidgranulomatosis, primary effusion lymphoma, intravascular large B celllymphoma, mediastinal large B cell lymphoma, heavy chain diseases(including γ, μ, and α disease), lymphomas induced by therapy withimmunosuppressive agents, such as cyclosporine-induced lymphoma, andmethotrexate-induced lymphoma.

In one embodiment of the present invention, the disorder involving cellsexpressing CD38 is Hodgkin's lymphoma.

Other examples of disorders involving cells expressing CD38 includemalignancies derived from T and NK cells including: mature T cell and NKcell neoplasms including T cell prolymphocytic leukemia, T cell largegranular lymphocytic leukemia, aggressive NK cell leukemia, adult T cellleukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type,enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma,subcutaneous panniculitis-like T cell lymphoma, blastic NK celllymphoma, Mycosis Fungoides/Sezary Syndrome, primary cutaneous CD30positive T cell lymphoproliferative disorders (primary cutaneousanaplastic large cell lymphoma C-ALCL, lymphomatoid papulosis,borderline lesions), angioimmunoblastic T cell lymphoma, peripheral Tcell lymphoma unspecified, and anaplastic large cell lymphoma.

Examples of malignancies derived from myeloid cells include acutemyeloid leukemia, including acute promyelocytic leukemia, and chronicmyeloproliferative diseases, including chronic myeloid leukemia.

In another embodiment of the present invention, the disorder involvingcells expressing CD38 is an immune disorder in which CD38 expressing Bcells, macrophages, plasma cells, monocytes and T cells are involved,such as an inflammatory and/or autoimmune disease. Examples of immunedisorders in which CD38 expressing B cells, plasma cells, monocytes andT cells are involved include autoimmune disorders, such as psoriasis,psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis,inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis,respiratory distress syndrome, meningitis, encephalitis, uveitis,glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesiondeficiency, multiple sclerosis, Raynaud's syndrome, Sjögren's syndrome,juvenile onset diabetes, Reiter's disease, Behçet's disease, immunecomplex nephritis, IgA nephropathy, IgM polyneuropathies,immune-mediated thrombocytopenias, such as acute idiopathicthrombocytopenic purpura and chronic idiopathic thrombocytopenicpurpura, hemolytic anemia, myasthenia gravis, lupus nephritis, systemiclupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis,pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener'sgranulomatosis, Omenn's syndrome, chronic renal failure, acuteinfectious mononucleosis, multiple sclerosis, HIV, and herpes virusassociated diseases. Further examples are severe acute respiratorydistress syndrome and choreoretinitis. Furthermore, other diseases anddisorders are included such as those caused by or mediated by infectionof B-cells with virus, such as Epstein-Barr virus (EBV).

In one embodiment, the disorder involving cells expressing CD38 isrheumatoid arthritis.

Further examples of inflammatory, immune and/or autoimmune disorders inwhich autoantibodies and/or excessive B and T lymphocyte activity areprominent and which may be treated according to the present inventioninclude the following: vasculitides and other vessel disorders, such asmicroscopic polyangiitis, Churg-Strauss syndrome, and otherANCA-associated vasculitides, polyarteritis nodosa, essentialcryoglobulinaemic vasculitis, cutaneous leukocytoclastic angiitis,Kawasaki disease, Takayasu arteritis, giant cell arthritis,Henoch-Schönlein purpura, primary or isolated cerebral angiitis,erythema nodosum, thrombangiitis obliterans, thrombotic thrombocytopenicpurpura (including hemolytic uremic syndrome), and secondaryvasculitides, including cutaneous leukocytoclastic vasculitis (e.g.,secondary to hepatitis B, hepatitis C, Waldenström's macroglobulinemia,B-cell neoplasias, rheumatoid arthritis, Sjögren's syndrome, or systemiclupus erythematosus); further examples are erythema nodosum, allergicvasculitis, panniculitis, Weber-Christian disease, purpurahyperglobulinaemica, and Buerger's disease; skin disorders, such ascontact dermatitis, linear IgA dermatosis, vitiligo, pyodermagangrenosum, epidermolysis bullosa acquisita, pemphigus vulgaris(including cicatricial pemphigoid and bullous pemphigoid), alopeciaareata (including alopecia universalis and alopecia totalis), dermatitisherpetiformis, erythema multiforme, and chronic autoimmune urticaria(including angioneurotic edema and urticarial vasculitis);immune-mediated cytopenias, such as autoimmune neutropenia, and pure redcell aplasia; connective tissue disorders, such as CNS lupus, discoidlupus erythematosus, CREST syndrome, mixed connective tissue disease,polymyositis/dermatomyositis, inclusion body myositis, secondaryamyloidosis, cryoglobulinemia type I and type II, fibromyalgia,phospholipid antibody syndrome, secondary hemophilia, relapsingpolychondritis, sarcoidosis, stiff man syndrome, and rheumatic fever; afurther example is eosinophil fasciitis; arthritides, such as ankylosingspondylitis, juvenile chronic arthritis, adult Still's disease, andSAPHO syndrome; further examples are sacroileitis, reactive arthritis,Still's disease, and gout; hematologic disorders, such as aplasticanemia, primary hemolytic anemia (including cold agglutinin syndrome),hemolytic anemia secondary to CLL or systemic lupus erythematosus; POEMSsyndrome, pernicious anemia, and Waldemström's purpurahyperglobulinaemica; further examples are agranulocytosis, autoimmuneneutropenia, Franklin's disease, Seligmann's disease, gamma heavy chaindisease, paraneoplastic syndrome secondary to thymoma and lymphomas, an,paraneoplastic syndrome secondary to thymoma and lymphomas, and factorVIII inhibitor formation; endocrinopathies, such as polyendocrinopathy,and Addison's disease; further examples are autoimmune hypoglycemia,autoimmune hypothyroidism, autoimmune insulin syndrome, de Quervain'sthyroiditis, and insulin receptor antibody-mediated insulin resistance;hepato-gastrointestinal disorders, such as celiac disease, Whipple'sdisease, primary biliary cirrhosis, chronic active hepatitis, andprimary sclerosing cholangiitis; a further example is autoimmunegastritis; nephropathies, such as rapid progressive glomerulonephritis,post-streptococcal nephritis, Goodpasture's syndrome, membranousglomerulonephritis, and cryoglobulinemic nephritis; a further example isminimal change disease; neurological disorders, such as autoimmuneneuropathies, mononeuritis multiplex, Lambert-Eaton's myasthenicsyndrome, Sydenham's chorea, tabes dorsalis, and Guillain-Barré'ssyndrome; further examples are myelopathy/tropical spastic paraparesis,myasthenia gravis, acute inflammatory demyelinating polyneuropathy, andchronic inflammatory demyelinating polyneuropathy; multiple sclerosis;cardiac and pulmonary disorders, such as COPD, fibrosing alveolitis,bronchiolitis obliterans, allergic aspergillosis, cystic fibrosis,Löffler's syndrome, myocarditis, and pericarditis; further examples arehypersensitivity pneumonitis, and paraneoplastic syndrome secondary tolung cancer; allergic disorders, such as bronchial asthma and hyper-IgEsyndrome; a further example is amaurosis fugax; ophthalmologicdisorders, such as idiopathic chorioretinitis; infectious diseases, suchas parvovirus B infection (including hands-and-socks syndrome);gynecological-obstretical disorders, such as recurrent abortion,recurrent fetal loss, and intrauterine growth retardation; a furtherexample is paraneoplastic syndrome secondary to gynaecologicalneoplasms; male reproductive disorders, such as paraneoplastic syndromesecondary to testicular neoplasms; and transplantation-deriveddisorders, such as allograft and xenograft rejection, andgraft-versus-host disease.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation.

The efficient dosages and the dosage regimens for the anti-CD38antibodies depend on the disease or condition to be treated and may bedetermined by the persons skilled in the art. An exemplary, non-limitingrange for a therapeutically effective amount of a compound of thepresent invention is about 0.005-100 mg/kg, such as 0.05-100 mg/kg or1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg,such as about 0.1-10 mg/kg, for instance about 0.1, 0.3, about 0.5,about 1, 2, 3, 4, 8, 16 or 24 mg/kg.

Administration may e.g. be intravenous, intramuscular, intraperitoneal,or subcutaneous, and for instance administered proximal to the site ofthe target. If desired, the effective daily dose of a pharmaceuticalcomposition may be administered as two, three, four, five, six or moresub-doses administered separately at appropriate intervals throughoutthe day, optionally, in unit dosage forms.

In one embodiment, the anti-CD38 antibodies may be administered byinfusion in a weekly dosage of from 10 to 500 mg/m², such as of from 200to 400 mg/m². Such administration may be repeated, e.g., 1 to 8 times,such as 3 to 5 times. The administration may be performed by continuousinfusion over a period of from 2 to 24 hours, such as of from 2 to 12hours.

In one embodiment, the anti-CD38 antibodies may be administered by slowcontinuous infusion over a long period, such as more than 24 hours, inorder to reduce toxic side effects.

In one embodiment the anti-CD38 antibodies may be administered in aweekly dosage of from 250 mg to 2000 mg, such as for example 300 mg, 500mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from4 to 6 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may e.g. be repeated one or more times as necessary, forexample, after 6 months or 12 months.

In one embodiment, the anti-CD38 antibodies may be administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

In another embodiment, the anti-CD38 antibodies may be administered by aregimen including one infusion of an anti-CD38 antibody of the presentinvention followed by an infusion of an anti-CD38 antibody of thepresent invention conjugated to a radioisotope. The regimen may berepeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of a compound of the present inventionin an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg,per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at leastone of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 after initiation of treatment, or any combination thereof,using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, orany combination thereof.

An “effective amount” for tumor therapy may also be measured by itsability to stabilize the progression of disease. The ability of acompound to inhibit cancer may be evaluated in an animal model systempredictive of efficacy in human tumors. Alternatively, this property ofa composition may be evaluated by examining the ability of the compoundto inhibit cell growth or to induce apoptosis by in vitro assays knownto the skilled practitioner. A therapeutically effective amount of atherapeutic compound may decrease tumor size, or otherwise amelioratesymptoms in a subject.

A “therapeutically effective amount” for rheumatoid arthritis may resultin an at least ACR₂₀ Preliminary Definition of Improvement in thepatients, such as in at least an ACR₅₀ Preliminary Definition ofImprovement, for instance at least an ARC₇₀ Preliminary Definition ofImprovement.

ACR₂₀ Preliminary Definition of Improvement is defined as:≥20% improvement in: Tender Joint Count (TJC) and Swollen Joint Count(SJC)and ≥20% improvement in 3 of following 5 assessments: Patient PainAssessment (VAS), Patient Global assessment (VAS), Physician GlobalAssessment (VAS), Patent Self-Assessed Disability (HAQ), Acute PhaseReactant (CRP or ESR). ACR₅₀ and ACR₇₀ are defined in the same way with≥50% and ≥70% improvements, respectively. For further details see Felsonet al., in American College of Rheumatology Preliminary Definition ofImprovement in Rheumatoid Arthritis; Arthritis Rheumatism 38, 727-735(1995).

Alternatively, a therapeutically effective amount for rheumatoidarthritis can be measured by DAS (disease activity score), includingDAS28 and/or DAS56, as defined by EU LAR.

An anti-CD38 antibody may also be administered prophylactically in orderto reduce the risk of developing cancer, delay the onset of theoccurrence of an event in cancer progression, and/or reduce the risk ofrecurrence when a cancer is in remission. This may be especially usefulin patients wherein it is difficult to locate a tumor that is known tobe present due to other biological factors.

Combination Therapy

The anti-CD38 antibodies of the present invention may also beadministered in combination therapy, i.e., combined with othertherapeutic agents relevant for the disease or condition to be treated.Such administration may be simultaneous, separate or sequential. Forsimultaneous administration the agents may be administered as onecompositions or as separate compositions, as appropriate.

Accordingly, the present invention provides methods for treating adisorder involving cells expressing CD38 as described above, whichmethods comprise administration of an anti-CD38 antibody of the presentinvention combined with one or more additional therapeutic agents asdescribed below.

In an embodiment of the invention the antibodies of the presentinvention are administered as a combination with other anti-CD38antibodies. Such antibodies are described in the present invention andin prior art. Specifically, antibodies are described in WO2006099875.More specifically, a combination of the present anti-CD38 antibodieswith anti-CD38 antibodies which are non-cross-blocking, such as antibody003 described in WO2006099875 are embodiments of the present invention.

In one embodiment, the combination therapy may include administration ofa composition of the present invention together with at least onecytotoxic agent, at least one chemotherapeutic agent, at least oneanti-angiogenic agent, at least one anti-inflammatory agent, and/or atleast one immunosuppressive and/or immunomodulatory agent.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing CD38, such as cancer, in asubject, which method comprises administration of a therapeuticallyeffective amount of an anti-CD38 antibody of the present invention andat least one chemotherapeutic agent to a subject in need thereof

In one embodiment, the present invention provides a method for treatingmultiple myeloma, which method comprises administration of atherapeutically effective amount of an anti-CD38 antibody of the presentinvention and at least one chemotherapeutic agent to a subject in needthereof.

In one embodiment, such a chemotherapeutic agent may be selected from anantimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea,asparaginase, gemcitabine, cladribine and similar agents.

In one embodiment, such a chemotherapeutic agent may be selected from analkylating agent, such as mechlorethamine, thioepa, chlorambucil,melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide,busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC),procarbazine, mitomycin C, cisplatin and other platinum derivatives,such as carboplatin, and similar agents.

In one embodiment, such a chemotherapeutic agent may be selected from anantibiotic, such as dactinomycin (formerly actinomycin), bleomycin,daunorubicin (formerly daunomycin), doxorubicin, idarubicin,mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC) andsimilar agents.

In one embodiment, such a chemotherapeutic agent may be selected from ananti-mitotic agent, such as taxanes, for instance docetaxel, andpaclitaxel, and vinca alkaloids, for instance vindesine, vincristine,vinblastine, and vinorelbine.

In one embodiment, such a chemotherapeutic agent may be selected from atopoisomerase inhibitor, such as topotecan.

In one embodiment, such a chemotherapeutic agent may be selected from agrowth factor inhibitor, such as an inhibitor of ErbB1 (EGFR) (such asgefitinib (Iressa®), cetuximab (Erbitux®), erlotinib (Tarceva®),HuMax-EGFr (zalutumumab, 2F8 disclosed in WO 2002/100348) and similaragents), an inhibitor of ErbB2 (Her2/neu) (such as trastuzumab(Herceptin®) and similar agents) and similar agents. In one embodiment,such a growth factor inhibitor may be a farnesyl transferase inhibitor,such as SCH-66336 and R115777. In one embodiment, such a growth factorinhibitor may be a vascular endothelial growth factor (VEGF) inhibitor,such as bevacizumab (Avastin®).

In one embodiment, such a chemotherapeutic agent may be a tyrosinekinase inhibitor, such as imatinib (Glivec, Gleevec ST1571), lapatinib,PTK787/ZK222584 and similar agents.

In one embodiment, such a chemotherapeutic agent may be a histonedeacetylase inhibitor. Examples of such histone deacetylase inhibitorsinclude hydroxamic acid-based hybrid polar compounds, such as SAHA(suberoylanilide hydroxamic acid).

In one embodiment, such a chemotherapeutic agent may be a P38a MAPkinase inhibitor, such as SCIO-469.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing CD38 in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-CD38 antibody of the present invention and at least one inhibitorof angiogenesis, neovascularization, and/or other vascularization to asubject in need thereof

In one embodiment, the present invention provides a method for treatingmultiple myeloma, which method comprises administration of atherapeutically effective amount of an anti-CD38 antibody of the presentinvention and at least one inhibitor of angiogenesis,neovascularization, and/or other vascularization to a subject in needthereof.

Examples of such angiogenesis inhibitors are urokinase inhibitors,matrix metalloprotease inhibitors (such as marimastat, neovastat, BAY12-9566, AG 3340, BMS-275291 and similar agents), inhibitors ofendothelial cell migration and proliferation (such as TNP-470,squalamine, 2-methoxyestradiol, combretastatins, endostatin,angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison,N.J.), R115777 (Janssen Pharmaceutica, Inc, Titusville, N.J.) andsimilar agents), antagonists of angiogenic growth factors (such asZD6474, SU6668, antibodies against angiogenic agents and/or theirreceptors (such as VEGF, bFGF, and angiopoietin-1), thalidomide(Thalomid®), thalidomide analogs (such as CC-5013 (lenalidomide,Revlimid™) and CC4047 (Actimid™), Sugen 5416, SU5402, antiangiogenicribozyme (such as angiozyme), interferon α (such as interferon α2a),suramin and similar agents), VEGF-R kinase inhibitors and otheranti-angiogenic tyrosine kinase inhibitors (such as SU011248),inhibitors of endothelial-specific integrin/survival signaling (such asvitaxin and similar agents), copper antagonists/chelators (such astetrathiomolybdate, captopril and similar agents), carboxyamido-triazole(CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E as wellas nucleotide molecules inhibiting angiogenesis (such asantisense-VEGF-cDNA, cDNA coding for angiostatin, cDNA coding for p53and cDNA coding for deficient VEGF receptor-2) and similar agents.

Other examples of such inhibitors of angiogenesis, neovascularization,and/or other vascularization are anti-angiogenic heparin derivatives andrelated molecules (e.g., heperinase III), temozolomide, NK4, macrophagemigration inhibitory factor (MIF), cyclooxygenase-2 inhibitors,inhibitors of hypoxia-inducible factor 1, anti-angiogenic soyisoflavones, oltipraz, fumagillin and analogs thereof, somatostatinanalogues, pentosan polysulfate, tecogalan sodium, dalteparin,tumstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin,antibodies against other relevant targets (such as anti-alpha-v/beta-3integrin and anti-kininostatin mAbs) and similar agents.

In one embodiment, the present invention provides the use of ananti-CD38 antibody of the present invention for the preparation of apharmaceutical composition to be administered with thalidomide(Thalomid®), thalidomide analogs (such as CC-5013 (lenalidomide,Revlimid™) and/or CC4047 (Actimid™). In a further embodiment, thepresent invention provides the use of an anti-CD38 antibody of thepresent invention for the preparation of a pharmaceutical composition tobe administered with thalidomide.

In one embodiment, the present invention provides the use of ananti-CD38 antibody of the present invention for the preparation of apharmaceutical composition to be administered with an anti-CD20antibody, such as rituximab (Rituxan®, Mabthera®), a human monoclonalanti-CD20 antibody as disclosed in WO 2004/035607, such as 11B8, 2F2(ofatumumab, Arzerra®) or 7D8.

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be a proteasome inhibitor, such as bortezomib(Velcade®).

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be a corticosteroid, such as prednisone,prednisolone, dexamethasone, etc.

In one embodiment, the anti-CD38 antibody of the present invention isused in combination with lenalidomide and dexamethasone for treating thedisorders as described above, such as multiple myeloma, e.g. relapsedmultiple myeloma.

In one embodiment, the anti-CD38 antibody of the present invention isused in combination with bortezomib and dexamethasone for treating thedisorders as described above, such as multiple myeloma, e.g. relapsedmultiple myeloma.

In one embodiment, the anti-CD38 antibody of the present invention isused in combination with bortezomib and prednisolone for treating thedisorders as described above, such as multiple myeloma, e.g. relapsedmultiple myeloma.

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be an anti-cancer immunogen, such as a cancerantigen/tumor-associated antigen (e.g., epithelial cell adhesionmolecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen(CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A,MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (e.g., humanpapillomavirus vaccines), tumor-derived heat shock proteins, and similaragents. A number of other suitable cancer antigens/tumor-associatedantigens described elsewhere herein and similar molecules known in theart may also or alternatively be used in such embodiment. Anti-cancerimmunogenic peptides also include anti-idiotypic “vaccines” such as BEC2anti-idiotypic antibodies, Mitumomab, CeaVac and related anti-idiotypicantibodies, anti-idiotypic antibody to MG7 antibody, and otheranti-cancer anti-idiotypic antibodies (see for instance Birebent et al.,Vaccine. 21(15), 1601-12 (2003), Li et al., Chin Med J (Engl). 114(9),962-6 (2001), Schmitt et al., Hybridoma. 13(5), 389-96 (1994), Maloneyet al., Hybridoma. 4(3), 191-209 (1985), Raychardhuri et al., J Immunol.137(5), 1743-9 (1986), Pohl et al., Int J Cancer. 50(6), 958-67 (1992),Bohlen et al., Cytokines Mol Ther. 2(4), 231-8 (1996) and Maruyama, JImmunol Methods. 264(1-2), 121-33 (2002)). Such anti-idiotypicAntibodies may optionally be conjugated to a carrier, which may be asynthetic (typically inert) molecule carrier, a protein (for instancekeyhole limpet hemocyanin (KLH) (see for instance Ochi et al., Eur JImmunol. 17(11), 1645-8 (1987)), or a cell (for instance a red bloodcell—see for instance Wi et al., J Immunol Methods. 122(2), 227-34(1989)).

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be a bisphosphonate. Examples of potentiallysuitable biphosphonates are pamidronate (Aredia®), zoledronic acid(Zometa®), clodronate (Bonefos®), risendronate (Actonel®), ibandronate(Boniva®), etidronate (Didronel®), alendronate (Fosamax®), tiludronate(Skelid®), incadronate (Yamanouchi Pharmaceutical) and minodronate(YM529, Yamanouchi).

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be a colony stimulating factor. Examples ofsuitable colony stimulating factors are granulocyte-colony stimulatingfactors (G-CSF), such as filgrastim (Neupogen®) and pegfilgrastim(Neulasta®), and granulocyte macrophage-colony stimulating factors(GM-CSF) such as sargramostim (Leukine®).

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be a erythropoietic agent. Examples of suitableerythropoietic agents are erythropoietin (EPO), such as epoetin alfa(for instance Procrit®, Epogen®, and Eprex®) and epoetin beta (forinstance NeoRecormon®) and erythropoiesis-stimulating proteins (forinstance Aranesp®).

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be an anti-cancer cytokine, chemokine, orcombination thereof. Examples of suitable cytokines and growth factorsinclude IFNγ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18,IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα (e.g., INFα2b),IFNβ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα.Suitable chemokines may include Glu-Leu-Arg (ELR)-negative chemokinessuch as IP-10, MCP-3, MIG, and SDF-1α from the human CXC and C-Cchemokine families. Suitable cytokines include cytokine derivatives,cytokine variants, cytokine fragments, and cytokine fusion proteins.These and other methods or uses involving naturally occurringpeptide-encoding nucleic acids herein may alternatively or additionallybe performed by “gene activation” and homologous recombination geneupregulation techniques, such as are described in U.S. Pat. Nos.5,968,502, 6,063,630 and 6,187,305 and EP 0505500.

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be an agent that modulates, e.g., enhances orinhibits, the expression or activity of Fcα or Fcγ receptors. Examplesof agents suitable for this use include interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-6 (IL-6), granulocytecolony-stimulating factor (G-CSF), such as filgrastim (Neupogen®) andpegfilgrastim (Neulasta®), and granulocyte macrophage-colony stimulatingfactors (GM-CSF) such as sargramostim (Leukine®), interferon-γ (IFN-γ),and tumor necrosis factor (TNF).

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be a cell cycle control/apoptosis regulator (or“regulating agent”). A cell cycle control/apoptosis regulator mayinclude molecules (i) that target and modulate cell cyclecontrol/apoptosis regulators such as cdc-25 (such as NSC 663284), (ii)cyclin-dependent kinases that overstimulate the cell cycle (such asflavopiridol (L868275, HMR1275), 7-hydroxy-staurosporine (UCN-01,KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerasemodulators (such as BIBR1532, SOT-095, GRN163 and compositions describedin for instance U.S. Pat. Nos. 6,440,735 and 6,713,055). Non-limitingexamples of molecules that interfere with apoptotic pathways includeTNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand(Apo-2L), agents inducing NF-κB blockade leading to inhibition of IL-6production, antibodies that activate TRAIL receptors, IFNs, anti-senseBcl-2, and As₂O₃ (arsenic trioxide, Trisenox®).

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be a hormonal regulating agent, such as agentsuseful for anti-androgen and anti-estrogen therapy. Examples of suchhormonal regulating agents are tamoxifen, idoxifene, fulvestrant,droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinylestradiol/estinyl, an antiandrogene (such as flutaminde/eulexin), aprogestin (such as such as hydroxy-progesterone caproate,medroxyprogesterone/provera, megestrol acepate/megace), anadrenocorticosteroid (such as hydrocortisone, prednisone), luteinizinghormone-releasing hormone (and analogs thereof and other LHRH agonistssuch as buserelin and goserelin), an aromatase inhibitor (such asanastrazole/arimidex, aminoglutethimide/cytraden, exemestane), a hormoneinhibitor (such as octreotide/sandostatin) and similar agents.

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be an anti-anergic agents (for instance smallmolecule compounds, proteins, glycoproteins, or antibodies that breaktolerance to tumor and cancer antigens). Examples of such compounds aremolecules that block the activity of CTLA-4, such as MDX-010 (Phan etal., PNAS USA 100, 8372 (2003)).

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be a tumor suppressor gene-containing nucleicacid or vector such as a replication-deficient adenovirus encoding humanrecombinant wild-type p53/SCH58500, etc.; antisense nucleic acidstargeted to oncogenes, mutated, or deregulated genes; or siRNA targetedto mutated or deregulated genes. Examples of tumor suppressor targetsinclude, for example, BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1, andDCC.

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be an anti-cancer nucleic acid, such as genasense(augmerosen/G3139), LY900003 (ISIS 3521), ISIS 2503, OGX-011 (ISIS112989), LE-AON/LEraf-AON (liposome encapsulated c-raf antisenseoligonucleotide/ISIS-5132), MG98, and other antisense nucleic acids thattarget PKCα, clusterin, IGFBPs, protein kinase A, cyclin D1, or Bcl-2h.

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be an anti-cancer inhibitory RNA molecule (seefor instance Lin et al., Curr Cancer Drug Targets. 1(3), 241-7 (2001),Erratum in: Curr Cancer Drug Targets. 3(3), 237 (2003), Lima et al.,Cancer Gene Ther. 11(5), 309-16 (2004), Grzmil et al., Int J Oncol.4(1), 97-105 (2004), Collis et al., Int J Radiat Oncol Biol Phys. 57(2Suppl), S144 (2003), Yang et al., Oncogene. 22(36), 5694-701 (2003) andZhang et al., Biochem Biophys Res Commun. 303(4), 1169-78 (2003)).

Compositions and combination administration methods of the presentinvention also include the administration of nucleic acid vaccines, suchas naked DNA vaccines encoding such cancer antigens/tumor-associatedantigens (see for instance U.S. Pat. Nos. 5,589,466, 5,593,972,5,703,057, 5,879,687, 6,235,523, and 6,387,888). In one embodiment, thecombination administration method and/or combination compositioncomprises an autologous vaccine composition. In one embodiment, thecombination composition and/or combination administration methodcomprises a whole cell vaccine or cytokine-expressing cell (for instancea recombinant IL-2 expressing fibroblast, recombinantcytokine-expressing dendritic cell, and the like) (see for instanceKowalczyk et al., Acta Biochim Pol. 50(3), 613-24 (2003), Reilly et al.,Methods Mol Med. 69, 233-57 (2002) and Tirapu et al., Curr Gene Ther.2(1), 79-89 (2002). Another example of such an autologous cell approachthat may be useful in combination methods of the present invention isthe MyVax® Personalized Immunotherapy method (previously referred to asGTOP-99) (Genitope Corporation—Redwood City, Calif., USA).

In one embodiment, the present invention provides combinationcompositions and combination administration methods wherein an anti-CD38antibody is combined or co-administered with an oncolytic virus.

Combination compositions and combination administration methods of thepresent invention may also involve “whole cell and “adoptive”immunotherapy methods. For instance, such methods may comprise infusionor re-infusion of immune system cells (for instance tumor-infiltratinglymphocytes (TILs), such as CD4⁺ and/or CD8⁺ T cells (for instance Tcells expanded with tumor-specific antigens and/or geneticenhancements), antibody-expressing B cells or other antibodyproducing/presenting cells, dendritic cells (e.g., anti-cytokineexpressing recombinant dendritic cells, dendritic cells cultured with aDC-expanding agent such as GM-CSF and/or Flt3-L, and/or tumor-associatedantigen-loaded dendritic cells), anti-tumor NK cells, so-called hybridcells, or combinations thereof. Cell lysates may also be useful in suchmethods and compositions. Cellular “vaccines” in clinical trials thatmay be useful in such aspects include Canvaxin™, APC-8015 (Dendreon),HSPPC-96 (Antigenics), and Melacine® cell lysates. Antigens shed fromcancer cells, and mixtures thereof (see for instance Bystryn et al.,Clinical Cancer Research Vol. 7, 1882-1887, July 2001), optionallyadmixed with adjuvants such as alum, may also be components in suchmethods and combination compositions.

In one embodiment, an anti-CD38 antibody of the present invention may bedelivered to a patient in combination with the application of aninternal vaccination method. Internal vaccination refers to inducedtumor or cancer cell death, such as drug-induced or radiation-inducedcell death of tumor cells, in a patient, that typically leads toelicitation of an immune response directed towards (i) the tumor cellsas a whole or (ii) parts of the tumor cells including (a) secretedproteins, glycoproteins or other products, (b) membrane-associatedproteins or glycoproteins or other components associated with orinserted in membranes, and/or (c) intracellular proteins or otherintracellular components. An internal vaccination-induced immuneresponse may be humoral (i.e. antibody—complement-mediated) orcell-mediated (e.g., the development and/or increase of endogenouscytotoxic T lymphocytes that recognize the internally killed tumor cellsor parts thereof). In addition to radiotherapy, non-limiting examples ofdrugs and agents that may be used to induce said tumor cell-death andinternal vaccination are conventional chemotherapeutic agents,cell-cycle inhibitors, anti-angiogenesis drugs, monoclonal antibodies,apoptosis-inducing agents, and signal transduction inhibitors.

Examples of other anti-cancer agents, which may be relevant astherapeutic agents for use in combination with the anti-CD38 antibody ofthe present invention for treating the disorders as described above aredifferentiation inducing agents, retinoic acid and retinoic acidanalogues (such as all trans retinoic acid, 13-cis retinoic acid andsimilar agents), vitamin D analogues (such as seocalcitol and similaragents), inhibitors of ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa,PDGFRbeta, Flk2, Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC, c-met,Ron, Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7 and similar agents.

Examples of other anti-cancer agents, which may be relevant astherapeutic agents for use in combination with the anti-CD38 antibody ofthe present invention for treating the disorders as described above arecathepsin B, modulators of cathepsin D dehydrogenase activity,glutathione-S-transferase (such as glutacylcysteine synthetase andlactate dehydrogenase), estramustine, epirubicin, HSP90 inhibitor like17-allyl amino geld-anamycin, antibodies directed against a tumorantigen such as PSA, CA125, KSA, etc., integrins like integrin β1,inhibitors of VCAM and similar agents.

Examples of other anti-cancer agents, which may be relevant astherapeutic agents for use in combination with the anti-CD38 antibodiesof the present invention for treating the disorders as described aboveare calcineurin-inhibitors (such as valspodar, PSC 833 and other MDR-1or p-glycoprotein inhibitors), TOR-inhibitors (such as sirolimus,everolimus and rapamcyin). and inhibitors of “lymphocyte homing”mechanisms (such as FTY720), and agents with effects on cell signalingsuch as adhesion molecule inhibitors (for instance anti-LFA, etc.).

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing CD38, such as cancer, in asubject, which method comprises administration of a therapeuticallyeffective amount of an anti-CD38 antibody of the present invention andradiotherapy to a subject in need thereof.

In one embodiment, the present invention provides a method for treatingmultiple myeloma, which method comprises administration of atherapeutically effective amount of an anti-CD38 antibody of the presentinvention and radiotherapy to a subject in need thereof.

Radiotherapy may comprise radiation or associated administration ofradiopharmaceuticals to a patient is provided. The source of radiationmay be either external or internal to the patient being treated(radiation treatment may, for example, be in the form of external beamradiation therapy (EBRT), brachytherapy (BT) or skeletal targetedradiotherapy). Radioactive elements that may be used in practicing suchmethods include, e.g., radium, cesium-137, iridium-192, americium-241,gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131,and indium-111.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing CD38 in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-CD38 antibody of the present invention to a subject in need thereofcombined with autologous peripheral stem cell or bone marrowtransplantation.

In one embodiment, the present invention provides a method for treatingmultiple myeloma, which method comprises administration of atherapeutically effective amount of an anti-CD38 antibody of the presentinvention to a subject in need thereof combined with autologousperipheral stem cell or bone marrow transplantation.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing CD38 in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-CD38 antibody of the present invention to a subject in need thereofcombined with orthopedic intervention.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing CD38 in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-CD38 antibody of the present invention and at least oneanti-inflammatory agent to a subject in need thereof.

In one embodiment such an anti-inflammatory agent may be selected from asteroidal drug and a NSAID (nonsteroidal anti-inflammatory drug).

In one embodiment such an anti-inflammatory agent may be selected fromaspirin and other salicylates, Cox-2 inhibitors (such as rofecoxib andcelecoxib), NSAIDs (such as ibuprofen, fenoprofen, naproxen, sulindac,diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac,oxaprozin, and indomethacin), anti-IL6R antibodies, anti-IL8 antibodies(e.g. antibodies described in WO2004058797, e.g. 10F8), anti-IL15antibodies (e.g. antibodies described in WO03017935 and WO2004076620),anti-IL15R antibodies, anti-CD4 antibodies (e.g. zanolimumab),anti-CD11a antibodies (e.g., efalizumab), anti-alpha-4/beta-1 integrin(VLA4) antibodies (e.g. natalizumab), CTLA4-Ig for the treatment ofinflammatory diseases, prednisolone, prednisone, disease modifyingantirheumatic drugs (DMARDs) such as methotrexate, hydroxychloroquine,sulfasalazine, pyrimidine synthesis inhibitors (such as leflunomide),IL-1 receptor blocking agents (such as anakinra), TNF-α blocking agents(such as etanercept, infliximab, and adalimumab) and similar agents.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing CD38 in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-CD38 antibody of the present invention and at least oneimmunosuppressive and/or immunomodulatory agent to a subject in needthereof.

In one embodiment, such an immunosuppressive and/or immunomodulatoryagent may be selected from cyclosporine, azathioprine, mycophenolicacid, mycophenolate mofetil, corticosteroids such as prednisone,methotrexate, gold salts, sulfasalazine, antimalarials, brequinar,leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine,cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyteglobulin, thymopentin, thymosin-α and similar agents.

In one embodiment, such an immunosuppressive and/or immunomodulatoryagent may be selected from immunosuppressive antibodies, such asantibodies binding to p75 of the IL-2 receptor, or antibodies binding tofor instance MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFNγ, TNF-α,IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11a, or CD58,or antibodies binding to their ligands.

In one embodiment, such an immunosuppressive and/or immunomodulatoryagent may be selected from soluble IL-15R, IL-10, B7 molecules (B7-1,B7-2, variants thereof, and fragments thereof), ICOS, and OX40, aninhibitor of a negative T cell regulator (such as an antibody againstCTLA4) and similar agents.

In one embodiment, the anti-CD38 antibody of the present invention maybe administered in combination with two or more immunosuppressive and/orimmunomodulatory agents, such as in combination with prednisone andcyclosporine; prednisone, cyclosporine and azathioprine; or prednisone,cyclosporine and mycophenolate mofetil.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing CD38 in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-CD38 antibody of the present invention and an anti-C3b(i) antibodyto a subject in need thereof.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing CD38 in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-CD38 antibody of the present invention and an anti-CD32b antibodyto a subject in need thereof. In one embodiment of the presentinvention, the anti-CD32b antibody is selected from HuMab-016, -020,-022, -024, 026, 028, -034, -038 or -053 all disclosed in WO2009/083009.

In one embodiment, a therapeutic agent for use in combination with theanti-CD38 antibody of the present invention for treating the disordersas described above may be selected from histone deacetylase inhibitors(for instance phenylbutyrate) and/or DNA repair agents (for instance DNArepair enzymes and related compositions such as dimericine).

Methods of the present invention for treating a disorder as describedabove comprising administration of a therapeutically effective amount ofan anti-CD38 antibody of the present invention may also compriseanti-cancer directed photodynamic therapy (for instance anti-cancerlaser therapy—which optionally may be practiced with the use ofphotosensitizing agent, see, for instance Zhang et al., J ControlRelease. 93(2), 141-50 (2003)), anti-cancer sound-wave and shock-wavetherapies (see for instance Kambe et al., Hum Cell. 10(1), 87-94(1997)), and/or anti-cancer nutraceutical therapy (see for instanceRoudebush et al., Vet Clin North Am Small Anim Pract. 34(1), 249-69,viii (2004) and Rafi, Nutrition. 20(1), 78-82 (2004). Likewise, ananti-CD38 antibody of the present invention may be used for thepreparation of a pharmaceutical composition for treating a disorder asdescribed above to be administered with anti-cancer directedphotodynamic therapy (for instance anti-cancer laser therapy—whichoptionally may be practiced with the use of photosensitizing agent,anti-cancer sound-wave and shock-wave therapies, and/or anti-cancernutraceutical therapy.

In a further embodiment, the anti-CD38 antibody of the present inventionis administered together with complement.

As described above, a pharmaceutical composition of the presentinvention may be administered in combination therapy, i.e., combinedwith one or more agents relevant for the disease or condition to betreated either as separate pharmaceutical compositions or with acompound of the present invention coformulated with one or moreadditional therapeutic agents as described above. Such combinationtherapies may require lower dosages of the compound of the presentinvention and/or the co-administered agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.

In one embodiment, the present invention provides a method for treatinga disorder involving cells expressing CD38 in a subject, which methodcomprises administration of a therapeutically effective amount of ananti-CD38 antibody of the present invention and at least oneimmunosuppressive and/or immunomodulatory agent to a subject in needthereof

Diagnostic Uses

The anti-CD38 antibodies of the invention may also be used fordiagnostic purposes. Thus, in a further aspect, the invention relates toa diagnostic composition comprising an anti-CD38 antibody as definedherein.

In one embodiment, the anti-CD38 antibodies of the present invention maybe used in vivo or in vitro for diagnosing diseases wherein activatedcells expressing CD38 play an active role in the pathogenesis, bydetecting levels of CD38, or levels of cells which contain CD38 on theirmembrane surface. This may be achieved, for example, by contacting asample to be tested, optionally along with a control sample, with theanti-CD38 antibody under conditions that allow for formation of acomplex between the antibody and CD38. Complex formation is thendetected (e.g., using an ELISA). When using a control sample along withthe test sample, complex is detected in both samples and anystatistically significant difference in the formation of complexesbetween the samples is indicative of the presence of CD38 in the testsample.

Thus, in a further aspect, the invention relates to a method fordetecting the presence of CD38 antigen, or a cell expressing CD38, in asample comprising:

-   -   contacting the sample with an anti-CD38 antibody of the        invention or a bispecific molecule of the invention, under        conditions that allow for formation of a complex between the        antibody and CD38; and    -   analyzing whether a complex has been formed.

In one embodiment, the method is performed in vitro.

More specifically, the present invention provides methods for theidentification of, and diagnosis of invasive cells and tissues, andother cells targeted by anti-CD38 antibodies of the present invention,and for the monitoring of the progress of therapeutic treatments, statusafter treatment, risk of developing cancer, cancer progression, and thelike.

In one example of such a diagnostic assay, the present inventionprovides a method of diagnosing the level of invasive cells in a tissuecomprising forming an immunocomplex between an anti-CD38 antibody andpotential CD38-containing tissues, and detecting formation of theimmunocomplex, wherein the formation of the immunocomplex correlateswith the presence of invasive cells in the tissue. The contacting may beperformed in vivo, using labeled isolated antibodies and standardimaging techniques, or may be performed in vitro on tissue samples.

Examples of conventional immunoassays provided by the present inventioninclude, without limitation, an ELISA, an RIA, FACS assays, plasmonresonance assays, chromatographic assays, tissue immunohistochemistry,Western blot, and/or immunoprecipitation using an anti-CD38 antibody.Suitable labels for the anti-CD38 antibody and/or secondary antibodiesused in such techniques include, without limitation, various enzymes,prosthetic groups, fluorescent materials, luminescent materials, andradioactive materials.

The anti-CD38 antibodies are particularly useful in the in vivo imagingof tumors. In vivo imaging of tumors associated with CD38 may beperformed by any suitable technique. For example, ⁹⁹Tc-labeling orlabeling with another gamma-ray emitting isotope may be used to labelanti-CD38 antibodies in tumors or secondary labeled (e.g., FITC labeled)anti-CD38 antibody:CD38 complexes from tumors and imaged with a gammascintillation camera (e.g., an Elscint Apex 409ECT device), typicallyusing low-energy, high resolution collimator or a low-energy all-purposecollimator. Stained tissues may then be assessed for radioactivitycounting as an indicator of the amount of CD38-associated peptides inthe tumor. The images obtained by the use of such techniques may be usedto assess biodistribution of CD38 in a patient, mammal, or tissue, forexample in the context of using CD38 or CD38-fragments as a biomarkerfor the presence of invasive cancer cells. Variations on this techniquemay include the use of magnetic resonance imaging (MRI) to improveimaging over gamma camera techniques. Similar immunoscintigraphy methodsand principles are described in, e.g., Srivastava (ed.), RadiolabeledMonoclonal Antibodies For Imaging And Therapy (Plenum Press 1988),Chase, “Medical Applications of Radioisotopes,” in Remington'sPharmaceutical Sciences, 18th Edition, Gennaro et al., (eds.), pp.624-652 (Mack Publishing Co., 1990), and Brown, “Clinical Use ofMonoclonal Antibodies,” in Biotechnology And Pharmacy 227-49, Pezzuto etal., (eds.) (Chapman & Hall 1993).

In a further aspect, the invention relates to a kit for detecting thepresence of CD38 antigen, or a cell expressing CD38, in a samplecomprising

-   -   an anti-CD38 antibody of the invention or a bispecific molecule        of the invention; and    -   instructions for use of the kit.

In one embodiment, the present invention provides a kit for diagnosis ofcancer comprising a container comprising an anti-CD38 antibody, and oneor more reagents for detecting binding of the anti-CD38 antibody to aCD38 peptide. Reagents may include, for example, fluorescent tags,enzymatic tags, or other detectable tags. The reagents may also includesecondary or tertiary antibodies or reagents for enzymatic reactions,wherein the enzymatic reactions produce a product that may bevisualized. In one embodiment, the present invention provides adiagnostic kit comprising one or more anti-CD38 antibodies of thepresent invention in labeled or unlabeled form in suitable container(s),reagents for the incubations for an indirect assay, and substrates orderivatizing agents for detection in such an assay, depending on thenature of the label. Control reagent(s) may also be included.

In a further aspect, the invention relates to an anti-idiotypic antibodywhich binds to an anti-CD38 antibody of the invention as describedherein.

An anti-idiotypic (Id) antibody is an antibody which recognizes uniquedeterminants generally associated with the antigen-binding site of anantibody. An Id antibody may be prepared by immunizing an animal of thesame species and genetic type as the source of an anti-CD38 mAb with themAb to which an anti-Id is being prepared. The immunized animaltypically can recognize and respond to the idiotypic determinants of theimmunizing antibody by producing an antibody to these idiotypicdeterminants (the anti-Id antibody). Such antibodies are described infor instance U.S. Pat. No. 4,699,880.

An anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. An anti-anti-Id may be epitopically identical tothe original mAb, which induced the anti-Id. Thus, by using antibodiesto the idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity. Anti-Idantibodies may be varied (thereby producing anti-Id antibody variants)and/or derivatized by any suitable technique, such as those describedelsewhere herein with respect to anti-CD38 antibodies of the presentinvention.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

EXAMPLES Example 1 Generation of Antibodies

HCo12 mice were immunized every fortnight with 20 μg purified HA-CD38alternating with NIH-3T3-CD38 transfected cells. The first immunizationwas performed with 5×10⁶ cells in 100 μl PBS, mixed with 100 μl CFA,i.p., the second and following immunizations with HA-CD38 s.c., in thepresence of 100 μl PBS, mixed with 100 μl IFA. The followingimmunizations with transfected cells were performed in the presence of200 μl PBS. After titer development, mice were boosted with 20 μgHA-CD38 in PBS, i.v.

Spleens were harvested from these mice, splenocytes were isolated andfused with PEG to a mouse myeloma cell line based upon standardprotocols. The resulting hybridomas were then screened for humanantibody production by ELISA and for CD38 specificity using humanCD38-transfected NS/0 cells by FACS analysis and recombinant HA-CD38protein binding by ELISA.

Sequence Analysis of the Anti-CD38 HuMab Variable Domains and Cloning inExpression Vectors

Total RNA of the anti-CD38 HuMabs was prepared from 5×10⁶ hybridomacells and 5′-RACE-Complementary DNA (cDNA) was prepared from 100 ngtotal RNA, using the SMART RACE cDNA Amplification kit (Clontech),according to the manufacturer's instructions.

VH and VL coding regions were amplified by PCR and cloned into thepCR-Blunt II-TOPO vector (Invitrogen) using the Zero Blunt PCR cloningkit (Invitrogen). For each HuMab, 16 VL clones and 8 VH clones weresequenced.

The VL and VH encoding regions were cloned into the pKappa and pG1fvectors.

CDR regions are indicated according to IMGT.

(http://imgt.cines.fr/IMGT_vquest/vquest?livret=0&Option=humanIg)The following IgG1,κ human monoclonal antibodies were identified:

VH VL 025 SEQ ID NO: 2  SEQ ID NO: 27 026 SEQ ID NO: 7  SEQ ID NO: 27028 SEQ ID NO: 12 SEQ ID NO: 37 049 SEQ ID NO: 17 SEQ ID NO: 42 056 SEQID NO: 22 SEQ ID NO: 47

Example 2 Electrospray Ionisation-Quadrupole-Time of Flight MassSpectrometry of Anti-CD38 Antibodies

Intact molecular weight data for anti-CD38 antibodies 025, 057 (sameamino acid sequence as antibody 026), 028, 049 and 056 were obtainedusing nanospray Electrospray-MS on a Q-TOF mass spectrometer. Aliquotsof each antibody sample were desalted offline using C₄ micro-tapcartridge and eluted in propanol/trifluoroacetic acid solvent. Theinstrument was calibrated using glu-fibrinopeptide fragment ions inMS/MS mode. MassLynx 4.0 software was used to de-convolute themultiply-charged data obtained.

Information on the molecular weight of light and heavy chain componentsof these antibodies was obtained following reduction usingdithiothreitol and analysis as described above.

TABLE 1 Mass of CD38 antibodies (in Dalton) Anti- intact MW Light HeavyChain body K0 K1 K2 Chain K0 K1 −025 144742.8 144874.1 144999.8 23357.849017.4 49145.8 −057 144828.2 144946.4 145071.2 23357.8 49047.4 49175.8−028 144818.3 144946.4 145074.7 23357.8 49054.5 49182.8 −049 144864.0144990.6 145117.8 23384.8 49049.3 49177.9 −056 145100.7 145222.7 23384.849099.5 49226.4

Example 3 Cross-Block Studies Using FACS

CHO-CD38 cells were incubated with an excess of unlabelled CD38-specificantibodies (4° C., 15 min). Then, cells were incubated with FITC-labeled005 antibody (concentration approximates EC₉₀, 4° C., 45 min) (005 isdisclosed in WO2006099875). After washing the cells twice with PBS-BSA,fluorescence was measured by flow cytometry. 005-FITC labeled antibodybinding was blocked by excess unlabelled antibodies 025, 026, 028, 049and 056, indicating that these antibodies have overlapping epitopes.Binding of 005-FITC was not blocked by excess unlabelled 003 (disclosedin WO2006099875) providing evidence for binding to a different epitope.

Cross-Blocking Studies Using ELISA

Soluble human CD38 was coated on the surface of an ELISA plate. CoatedCD38 was incubated with an excess of unlabelled CD38 specific antibodiesfor about 15 minutes and subsequently biotinylated CD38-specificantibodies were added (concentration approximates EC₉₀, RT, 1 hour).After washing three times with PBS/Tween, horseradish peroxidase(HRP)-conjugated streptavidine was added and the mixture was incubatedfor 1 hour at RT. The complex was detected by addition of anABTS-solution and the HRP mediated substrate conversion was measuredusing an ELISA reader at OD 405 nm.

Cross-Blocking Studies Using Sandwich-ELISA

Anti-CD38 antibodies were coated on the surface of an ELISA plate.Plate-bound antibodies were incubated with biotinylated soluble CD38 inthe presence of an excess of anti-CD38 antibodies in fluid phase. Afterwashing with PBS/Tween, bound biotinylated CD38 was detected withHRP-conjugated streptavidine for 1 hr at RT. This complex was detectedby addition of an ABTS-solution (after washing with PBS/Tween) and theHRP mediated substrate conversion was measured using an ELISA reader atOD 405 nm.

Example 4 Epitope Mapping Construction of HA-CD38 and His-CD38Expression Vectors.

The encoding sequences for the extracellular domain of human CD38(identical to amino acids 45-300 from Genbank entry AAA68482) wereamplified from plasmid pCIpuroCD38 (obtained from Prof. M. Glennie,Tenovus Research Laboratory, Southampton General Hospital, Southampton,UK) using PCR, introducing, restriction sites, an ideal Kozak sequence(GCCGCCACC), and sequences encoding a signal peptide and a N-terminal HAtag (ypydvpdya). The construct was cloned in the mammalian expressionvector pEE13.4 (Lonza Biologics). This construct was namedpEE13.4HACD38.

A similar construct was made synthetically and fully codon optimized(GeneArt, Regensburg, Germany), replacing the HA tag encoding part by aHis tag (HHHHHH) encoding part. The construct was cloned in pEE13.4 andnamed pEE13.4HisCD38.

Site Directed Mutagenesis

Several Mutations were Introduced in the Putative Antibody Binding Siteon the CD38 Molecule.

DNA substitutions leading to T237A, Q272R or S274F amino acidsubstitutions were generated using the QuickChange II XL Site-directedMutagenesis Kit (Stratagene, Amsterdam, The Netherlands) in thepEE13.4HACD38 vector. Similarly a D202G encoding substitution wasintroduced in the pEE13.4HisCD38 vector.

Transient Expression in HEK-293F Cells

Freestyle™ 293-F (a HEK-293 subclone adapted to suspension growth andchemically defined Freestyle medium, (HEK-293F)) cells were obtainedfrom Invitrogen and transfected with pEE13.4HACD38, pEE13.4HisCD38 orthe four constructs carrying the mutations, according to themanufacturer's protocol using 293fectin (Invitrogen). Cell culturesupernatants of transfected cells were used in ELISA for anti-CD38binding studies.

Anti-CD38 Antibody Binding

Mutations T237A, Q272R, and S274F: ELISA plates (Greiner, #655092) werecoated 0/N at 4° C. with 1 μg anti-HA antibody (Sigma, #H-9658) andsubsequently blocked with 2% chicken serum. Culture supernatants oftransfected HEK293F cells were diluted, applied to the ELISA plates andincubated for 1 hr at RT. After washing, serial dilutions of anti-CD38antibodies were added and incubated for 1 hr at RT. Bound antibodieswere detected with HRP-conjugated goat-anti-human IgG antibodies. Theassay was developed with ABTS (Roche, #1112597) and the absorbance wasmeasured at 405 nm using a spectrophotometer.

Mutation D202G: ELISA plates (Greiner, #655092) were coated 0/N at 4° C.with 1 μg penta-His (Qiagen #34660) and subsequently blocked with 2%PBS/BSA. Culture supernatants of transfected HEK293F cells were diluted,applied to the ELISA plates and incubated for 2 hr at RT. After washing,serial dilutions of anti-CD38 antibodies were added and incubated for 1hr at RT. Bound antibodies were detected with HRP-conjugatedgoat-anti-human IgG antibodies. The assay was developed with ABTS(Roche, #1112597) and the absorbance was measured at 405 nm using aspectrophotometer.

This study revealed that binding of 025, 026, 028 and 049 was notsensitive to mutations T237A, Q272R, S274F, and A199T, but was seriouslyaffected by D202G (025, 028, 049) (FIG. 2).

Example 5 Binding of Anti-CD38 Antibodies to CD38-Transfected CHO(CHO-CD38) Cells and to Daudi-Luc Cells

After harvesting and counting, Daudi-luc cells, CHO cells transfectedwith CD38 and control CHO cells, were resuspended in PBS (1×10⁶cells/mL). Cells were transferred to 96-well V-bottom plates (100μL/well) and washed twice in PBS-BSA (PBS supplemented with 0.1% BSA and0.02% Na-azide). 50 μL antibody in PBS-BSA was added in three-folddilutions ranging from 0.3 to 30 μg/mL (4° C., 30 min). After washingthree times in PBS-BSA, 50 μL (1:400 dilution) of rabbit anti-humanIgG-FITC in PBS-BSA was added (4° C. in the dark, 30 minutes). Cellswere washed three times and specific binding of CD38-antibodies toCHO-CD38 and Daudi-luc cells was detected by flow cytometry.

FIG. 3 shows that 025, 026, 028, 049, and 056 bind to CHO-CD38 cells andto Daudi-luc cells.

Example 6 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

The ability of anti-CD38 antibodies to perform ADCC of Daudi-luc cellswas determined as explained below. As effector cells, peripheral bloodmononuclear cells from healthy volunteers (UMC Utrecht, The Netherlands)were used.

Daudi-luc cells were collected (5×10⁶ cells) in RPMI⁺⁺ (RPMI 1640culture medium supplemented with 10% cosmic calf serum (HyClone, Logan,Utah, USA)), to which 100 μCi ⁵¹Cr (Chromium-51; Amersham BiosciencesEurope GmbH, Roosendaal, The Netherlands) was added, and the mixture wasincubated in a 37° C. water bath for 1 hr. After washing of the cells(twice in PBS, 1500 rpm, 5 min), the cells were resuspended in RPMI⁺⁺and counted by trypan blue exclusion. Cells were brought atconcentration of 1×10⁵ cells/mL.

Preparation of Effector Cells

Fresh peripheral blood mononuclear cells (healthy volunteers, UMCUtrecht, Utrecht, The Netherlands) were isolated from 40 ml of heparinblood by Ficoll (Bio Whittaker; lymphocyte separation medium, cat17-829E) according to the manufacturer's instructions. Afterresuspension of cells in RPMI⁺⁺, cells were counted by trypan blueexclusion and brought at concentration of 1×10⁷ cells/ml.

ADCC Set Up

50 μl of ⁵¹Cr-labeled targets cells were pipetted into 96-well plates,and 50 μl of antibody was added, diluted in RPMI⁺⁺ (final concentrations10, 1, 0.1, 0.01 μg/ml). Cells were incubated (RT, 15 min), and 50 μleffector cells were added, resulting in an effector to target ratio of100:1 (for determination of maximal lysis, 100 μl 5% Triton-X100 wasadded instead of effector cells; for determination of spontaneous lysis,50 μL target cells and 100 μL RPMI⁺⁺ were used). Cells were spun down(500 rpm, 5 min), and incubated (37° C., 5% CO₂, 4 hr). After spinningdown cells (1500 rpm, 5 min), 100 μL of supernatant was harvested intomicronic tubes, and counted in gamma counter. The percentage specificlysis was calculated as follows:

(cpm sample−cpm target cells only)/(cpm maximal lysis−cpm target cellsonly)

wherein cpm is counts per minute.025, 026, 028, 049, and 056 induced ADCC mediated lysis in Daudi cells(FIG. 4).

Example 7 Complement-Dependent Cytotoxicity (CDC)

After harvesting and counting of Daudi-luc cells, the viability of thecells should be ≥90%. After washing (PBS), cells are resuspended at2.0×10⁶ cells/ml in RPMI-B (RPMI supplemented with 1% BSA). Thereafter,cells are put in 96-well round-bottom plates at 1×10⁵ cells/well (50μL/well). Then, 50 μL antibodies is added to the wells (finalconcentration range between 0-100 μg/ml (three-fold dilutions inRPMI-B)). After incubation (RT, 15 min), 11 μL of pooled human serum(pool of 18 healthy donors) was added to each well (37° C., 45 min).Wells were resuspended once and 120 μL was transferred to FACS tubes(Greiner). Then, 10 μL propidium iodide (PI; Sigma-Aldrich Chemie B.V.)was added (10 μg/ml solution) to this suspension. Lysis was detected byflow cytometry (FACScalibur™, Becton Dickinson, San Diego, Calif., USA)by measurement of the percentage of dead cells (corresponds toPI-positive cells).

FIG. 5 presents CDC-mediated CHO-CD38 cell lysis caused by anti-CD38antibodies 025, 026, 028, 049 and 056. These anti-CD38 antibodies failedto induce CDC of Daudi-luc cells.

Example 8 Enzymatic Activity

The effects of anti-CD38 antibodies on the enzymatic activities of CD38were determined. CD38 is known to catalyze several different enzymaticreactions, including a cyclase reaction converting NAD into cyclic ADPribose (cADPR), a hydrolase reaction converting NAD or cADPR into ADPribose, and a base-exchange reaction in which nicotinic acid adeninedinucleotide 2′-phosphate (NAADP) is produced.

Cyclase Activity NGD Assay

The ability of anti-CD38 antibodies to interfere with the cyclaseactivity of CD38 using NGD as a substrate was measured in an assayessentially as described in Graeff et al., J. Biol. Chem. 269,30260-30267 (1994):

Briefly, substrate NOD⁺ (80 μM) was incubated with CD38 (0.6 μg/mlHis-tagged extracellular domain of human CD38, see Example 3 ofWO2006099875 regarding purification of His-CD38 in a buffer containing20 mM Tris-HCl, pH 7.0). The production of cGDPR can be monitoredspectrophotometrically at the emission wavelength of 410 nm (excitationat 300 nm). In this example an excitation filter of 340±60 nm and anemission filter of 430±8 nm were used.

To test the effect of 025, 026, 028, 049 and 056 on the enzymaticactivity of CD38, recombinant His-CD38 protein was pre-incubated for 15minutes at room temperature with 3 μg/ml of the antibodies before addingthe substrate NGD⁺. The production of cyclic GDP-ribose (cGDPR) wasrecorded after 90 minutes.

FIG. 6A shows that antibodies 025, 026, 028, 049 and 056 have apronounced inhibitory effect on the production of cGDPR. After 90minutes, 3 μg/ml of antibody (025, 026, 028, 049 or 056) resulted in a53-66% reduced production of cGDPR. In a time course experiment it wasshown that the rate of cGDPR production was reduced in samples treatedwith the CD38-specific antibody mAb 028 compared to the cGDPR productionin the presence of HuMab-KLH or in the untreated CD38 control (FIG. 6B).FIG. 6C shows a dose response curve (0.01-30 μg/ml) for antibody 028. Inthis experiment a maximum reduction of cGDPR production of 41% isobserved.

To test the effect of 028 on the enzymatic activity of cellularexpressed CD38, CHO-CDC38 cells were pre-incubated for 30 minutes atroom temperature with a serial dilution of 028 (0.01-30 μg/ml) beforeadding the substrate NGD⁺. The production of cyclic GDP-ribose (cGDPR)was recorded after 90 min. As shown in FIG. 6D antibody 028 inhibits theproduction of cGDPR in a concentration dependent fashion.

Reverse Cyclase Reaction

The effect of mAb 028 on cADPR production from NAD by CD38 wasdetermined by the reverse cyclase reaction. This assay is based on thereversibility of the reaction catalyzed by CD38. In the presence of highconcentrations of nicotinamide and cADPR, the ADP-ribosyl cyclases canproduce NAD. Antibodies were diluted to 10 μg/ml in 20 mM Tris-HCl,0.01% (v/v) BSA, pH 7.2 (Tris/BSA). Human recombinant CD38 was dilutedto 2 μg/ml with Tris/BSA. The antibodies were preincubated for 10minutes with CD38 by mixing equal volumes (50 μL) of the dilutedantibodies with the diluted CD38. The preincubation was done at roomtemperature. The reaction was initiated by transferring 25 μL ofCD38/antibody mixture to 25 μL of a solution containing 1 mM cADPR and10 mM nicotinamide. The reaction was allowed to proceed at roomtemperature for 1 to 20 minutes and was stopped at the appropriate timeby filtering the entire sample through a Millipore MultiScreen-IP Filter96-well plate to remove protein. The resulting NAD produced was measuredby the method of Graeff and Lee (1). Controls containing nicotinamidewithout cADPR were run to estimate the amount NAD contaminating thereagents. In these experiments there was undetectable contaminating NAD.

Table 2 shows that 1 μg/ml mAb-028 reduced cADPR production from NAD by67%. mAb-KLH had no effect on cADPR production from NAD.

TABLE 2 The effect of antibody 028 on cADPR production from NADCondition pmol cADPR/min CD38 control 4.3 mAb-KLH 4.3 mAb-028 1.48-amino-NAD (8NH2-NAD) Assay

As cADPR production only accounts for approximately 1% of the productgenerated from NAD by CD38 (ADPR accounts for the rest), ribosyl cyclaseactivity was also assessed using 8-amino-NAD (8NH2-NAD) as a substrate.Unlike NAD, a considerably larger amount (approximately 8%) of the8NH2-NAD substrate is cyclized to 8-amino-cADPR (8NH2-cADPR) and isdetectable by HPLC analysis. Briefly, antibodies were diluted to 10μg/mL in 20 mM Tris-HCl, 0.01% (v/v) BSA, pH 7.2 (Tris/BSA). Humanrecombinant CD38 was diluted to 2 μg/ml with Tris/BSA. The antibodieswere preincubated for 10 minutes with CD38 by mixing equal volumes (50μL) of the diluted antibodies with the diluted CD38. The preincubationwas done at room temperature. The reaction was initiated by transferring25 μL of CD38/antibody mixture to 75 μL of 0.5 mM 8NH2-NAD. The reactionwas allowed to proceed at room temperature for 10 minutes and wasstopped at the appropriate time by filtering the entire sample through aMillipore MultiScreen-IP Filter 96-well plate to remove protein. Thereaction products (8NH2-cADPR and 8NH2-ADPR) were analyzed by reversephase HPLC as follows. The column was a 0.46×15 cm LC18-T reverse phasecolumn from Supelco. Solvent A consisted of 20 mM KH2PO4, 5 mMtetrabutylammonium phosphate, pH 6 and solvent B consisted of 50% A and50% methanol. The flow rate was 1 mL/min and the initial composition ofsolvents was 15% B. Separation of substrates and products wasaccomplished using the following gradient: 0 to 3.5 minutes (15% B), 3.5to 5.5 minutes (15 to 32.5% B), 5.5 to 9 minutes (32.5 to 40% B), 9 to11.5 minutes (40 to 50% B) and 16 to 18 minutes (50 to 15% B) gradientwas used to elute the substrates and products. Samples were prepared byadding 400 μL of solvent A to 100 μL of filtered sample. The entiresample was injected. The flow rate and buffer composition werecontrolled by Beckman 125 HPLC pumps and System Gold software and peakswere detected with a Beckman 166 UV detector. The areas of the 8NH2-NAD,8NH2-cADPR and 8NH2-ADPR peaks were used to calculate the amount of8NH2-cADPR produced in the assay. The HPLC system is based on a systemdescribed by Schweitzer et al. (2).

FIG. 7A shows that mAb-028 inhibits 8NH2-cADPR by 78%. mAb-028 inhibits8NH2-cADPR production in a concentration dependent manner (FIG. 7B)

Thus mAb-028 inhibits the ADP-ribosyl cyclase reaction of CD38 asassayed by three different methods.

Hydrolase Activity Hydrolase Activity Analysis by HPLC

The hydrolase activity was measured by determining the amount of ADPRproduced from cADPR or NAD by HPLC. Antibodies were diluted to 10 μg/mLor titrated in 20 mM Tris-HCl, 0.01% (v/v) BSA, pH 7.2 (Tris/BSA). Humanrecombinant CD38 was diluted to 2 μg/mL with Tris/BSA. The antibodieswere preincubated for 10 minutes with CD38 by mixing equal volumes (50μL) of the diluted antibodies with the diluted CD38. The preincubationwas done at room temperature. For the HPLC-based method the cADPRhydrolase reaction was initiated by transferring 40 μL of CD38/antibodymixture to 10 μL of 4.3 mM cADPR and the NADase reaction was initiatedby transferring 40 μL of CD38/antibody mixture to 10 μL 1 mM NAD. Thereaction was allowed to proceed at room temperature and was stopped atthe appropriate time by adding 25 μL of 1 M HCl. Protein was removed byfiltering the entire sample through a Millipore MultiScreen-IP Filter96-well plate. Each filtrate was neutralized by adding 15 μL of 2MTris-base and kept on ice until analyzed by HPLC. The analysis ofhydrolase activity is based on the HPLC assay developed by Lee andAarhus (3). The samples were analyzed on a 0.5×5 cm column of AG MP-1(trifluoroacetate form) eluted at 3 mL/min with a 0 to 150 mM concaveupward gradient of trifluoroacetic acid (TFA) over 10 minutes. The flowrate and buffer composition were controlled by Beckman 125 HPLC pumpsand System Gold software and peaks were detected with a Beckman 166 UVdetector. The areas of NAD, cADPR and ADPR were used to calculate theamount of ADPR produced in the assay.

At concentrations of 10 μg/mL mAb-028, but not mAb-KLH, stimulated thecADPR hydrolase activity by 62% and the NAD hydrolase activity by 37%compared to the CD38 control (FIG. 8A). FIG. 8B shows that mAb-028stimulated cADPR hydrolysis in a dose-dependent manner. Atconcentrations of 30 μg/mL, mAb-028 stimulated hydrolase activity by78%.

Hydrolase Activity Analysis by Thin Layer Chromatography (TLC)

The hydrolase activity was measured by measuring the amount of ³²P-ADPRproduced from ³²P-cADPR by thin layer chromatography (4). The ³²P-basedcADPR hydrolase reaction was initiated by adding 20 μL of CD38/antibodymixture (as above) to 5 μL of a mixture containing 0.5 mM cADPR andapproximately 0.1 μCi of ³²P-cADPR. The reaction was allowed to proceedat room temperature and at the appropriate times, 5 μL of the reactionwas added to 5 μL of 150 mM TFA to stop the reaction. The reaction wasanalyzed by PEI-cellulose thin layer chromatography (TLC). One (1) μL ofeach stopped reaction sample was spotted on the origin of aPEI-cellulose TLC plate (10×20 cm). The plates were developed with 0.2 MNaCl in 30% (v/v) ethanol. The plates were dried and exposed tophosphoimage screens. The screens were analyzed on a Packard CyclonePhosphorimager to determine the amount of ³²P-ADPR produced.

FIG. 8C shows that mAb-028 stimulated ³²P-cADPR hydrolysis in adose-dependent manner. These results were similar to the results of thecADPR hydrolase activity measured by HPLC (see FIG. 8B).

Base-Exchange Activity

The effect of CD38 antibodies on nicotinic acid adenine dinucleotide2′-phosphate (NAADP) synthesis by the base-exchange activity of CD38 wasassessed. Antibodies (mAb-KLH and 028) were diluted to 40 μg/mL in 20 mMHepes, pH 7.3, 0.01% (v/v) BSA (Hepes/BSA). Human recombinant CD38 wasdiluted to 2 μg/mL with Hepes/BSA. The antibodies were preincubated for10 minutes with CD38 by mixing equal volumes (90 μL) of the dilutedantibodies with the diluted CD38. The preincubation was performed atroom temperature. The base-exchange reaction was initiated bytransferring 50 μL of CD38/antibody mixture to 50 μL of a reactionmixture containing 200 mM sodium acetate, pH 4.0, 25 mM nicotinic acidand 2 mM nicotinamide adenine dinucleotide 2′-phosphate (NADP). Thereaction was allowed to proceed at room temperature for 30 minutes andwas stopped by filtering the entire sample through a MilliporeMultiScreen-IP Filter 96-well plate to remove protein. The reactionproducts in the filtrates were determined by anion-exchange HPLC on a0.5×5 cm column of AG MP-1 (trifluoroacetate form) eluted at 1 mL/minwith a 0 to 150 mM concave upward gradient of trifluoroacetic acid (TFA)over 30 minutes (5). The filtrates (50 μL) were neutralized by adding 5μL of 2 M Tris-base just prior to injection. The flow rate and buffercomposition were controlled by Beckman 125 HPLC pumps and System Goldsoftware and peaks were detected with a Beckman 166 UV detector. Theareas of the NADP, NAADP (base-exchange product) and adenosinediphospho-ribose 2′-phosphate (ADPR-P, hydrolytic product) peaks wereused to calculate the rates of NAADP synthesis and NADP hydrolysis.

FIG. 9 shows that mAb-028 inhibits the ability of CD38 to catalyze theformation of NAADP. The inhibition of NAADP production by mAb-028 isconcentration dependent (FIG. 9B) with an IC50 of 0.14 μg/mL.

LIST OF REFERENCES

-   1. Graeff, R., and H. C. Lee. 2002. A novel cycling assay for    cellular cADP-ribose with nanomolar sensitivity. Biochem J    361:379-384.-   2. Schweitzer, K., G. W. Mayr, and A. H. Guse. 2001. Assay for    ADP-ribosyl cyclase by reverse-phase high-performance liquid    chromatography. Anal Biochem 299:218-226.-   3. Lee, H. C., and R. Aarhus. 1993. Wide distribution of an enzyme    that catalyzes the hydrolysis of cyclic ADP-ribose. Biochim Biophys    Acta 1164:68-74.-   4. White, T. A., S. Johnson, T. F. Walseth, H. C. Lee, R. M.    Graeff, C. B. Munshi, Y. S. Prakash, G. C. Sieck, and M. S.    Kannan. 2000. Subcellular localization of cyclic ADP-ribosyl cyclase    and cyclic ADP-ribose hydrolase activities in porcine airway smooth    muscle. Biochim Biophys Acta 1498:64-71.-   5. Aarhus, R., R. M. Graeff, D. M. Dickey, T. F. Walseth, and H. C.    Lee. 1995. ADP-ribosyl cyclase and CD38 catalyze the synthesis of a    calcium-mobilizing metabolite from NADP. J Biol Chem    270:30327-30333.

1-74. (canceled)
 75. A method for detecting the presence of CD38antigen, or a cell expressing CD38, in a sample comprising: (a)contacting the sample with an antibody that binds to human CD38 (SEQ IDNO: 52), wherein the antibody does not bind to a variant of human CD38wherein Asp in position 202 has been substituted with Gly to the samedegree that it binds to human CD38 under conditions that allow forformation of a complex between the antibody and CD38; and (b) analyzingwhether a complex has been formed.
 76. The method of claim 75, whereinthe sample is a human tissue sample.
 77. The method of claim 75, whichis performed in vitro.
 78. The method of claim 75, which is performed invivo.
 79. The method of claim 75, wherein the cell expressing CD38 is atumor cell.
 80. The method of claim 75, wherein the method is used todiagnose the level of invasive cells in a tissue, wherein the formationof a complex between the antibody and CD38 correlates with the presenceof invasive cells in the tissue.
 81. The method of claim 75, wherein theformation of the complex is detected using a method selected from thegroup consisting of: enzyme linked immunosorbent assay, RIA, FACS assay,plasma resonance assay, chromatography, tissue immunohistochemistry,Western blot, and immunoprecipitation.
 82. The method of claim 75,wherein the antibody comprises a detectable label selected form thegroup consisting of an enzyme, prosthetic group, fluorescent label,luminescent label, and radioactive label.
 83. The method of claim 75,wherein the formation of the complex is detected using a secondaryantibody comprising a detectable label.
 84. The method of claim 83,wherein the detectable label is selected from the group consisting of anenzyme, prosthetic group, fluorescent label, luminescent label, andradioactive label.
 85. The method of claim 75, wherein the antibodycomprises: (i) a VH CDR1 having the sequence as set forth in any of thesequences SEQ ID NOs: 3, 8, 13, 18, and 23, a VH CDR2 having thesequence as set forth in any of the sequences SEQ ID NOs: 4, 9, 14, 19,and 24, a VH CDR3 having the sequence as set forth in any of thesequences SEQ ID NOs: 5, 10, 15, 20, and 25, a VL CDR1 having thesequence as set forth in any of the sequences SEQ ID NOs: 28, 33, 38,43, and 48, a VL CDR2 having the sequence as set forth in any of thesequences SEQ ID NOs: 29, 34, 39, 44, and 49, a VL CDR3 having thesequence as set forth in any of the sequences SEQ ID NOs: 30, 35, 40,45, and 50, (ii) a VH CDR1 having the sequence as set forth in SEQ IDNO: 3, a VH CDR2 having the sequence as set forth in SEQ ID NO: 4, a VHCDR3 having the sequence as set forth in SEQ ID NO: 5, a VL CDR1 havingthe sequence as set forth in SEQ ID NO: 28, a VL CDR2 having thesequence as set forth in SEQ ID NO: 29, and a VL CDR3 having thesequence as set forth in SEQ ID NO: 30, (iii) a VH CDR1 having thesequence as set forth in SEQ ID NO: 8, a VH CDR2 having the sequence asset forth in SEQ ID NO: 9, a VH CDR3 having the sequence as set forth inSEQ ID NO: 10, a VL CDR1 having the sequence as set forth in SEQ ID NO:33, a VL CDR2 having the sequence as set forth in SEQ ID NO: 34, and aVL CDR3 having the sequence as set forth in SEQ ID NO: 35, (iv) a VHCDR1 having the sequence as set forth in SEQ ID NO: 13, a VH CDR2 havingthe sequence as set forth in SEQ ID NO: 14, a VH CDR3 having thesequence as set forth in SEQ ID NO: 15, a VL CDR1 having the sequence asset forth in SEQ ID NO: 38, a VL CDR2 having the sequence as set forthin SEQ ID NO: 39, and a VL CDR3 having the sequence as set forth in SEQID NO: 40, (v) a VH CDR1 having the sequence as set forth in SEQ ID NO:18, a VH CDR2 having the sequence as set forth in SEQ ID NO: 19, a VHCDR3 having the sequence as set forth in SEQ ID NO: 20, a VL CDR1 havingthe sequence as set forth in SEQ ID NO: 43, a VL CDR2 having thesequence as set forth in SEQ ID NO: 44, and a VL CDR3 having thesequence as set forth in SEQ ID NO: 45, (vi) a VH CDR1 having thesequence as set forth in SEQ ID NO: 23, a VH CDR2 having the sequence asset forth in SEQ ID NO: 24, a VH CDR3 having the sequence as set forthin SEQ ID NO: 25, a VL CDR1 having the sequence as set forth in SEQ IDNO: 48, a VL CDR2 having the sequence as set forth in SEQ ID NO: 49, anda VL CDR3 having the sequence as set forth in SEQ ID NO: 50, or (vii) avariant of any of the antibodies defined above, wherein the variantpreferably has at most 1, 2, or 3 amino acid modifications, morepreferably amino-acid substitutions, such as conservative amino acidsubstitutions in one or more of the sequences.
 86. The method of claim75, wherein the antibody comprises a VH CDR1 having the sequence as setforth in SEQ ID NO: 3, a VH CDR2 having the sequence as set forth in SEQID NO: 4, a VH CDR3 having the sequence as set forth in SEQ ID NO: 5, aVL CDR1 having the sequence as set forth in SEQ ID NO: 28, a VL CDR2having the sequence as set forth in SEQ ID NO: 29, and a VL CDR3 havingthe sequence as set forth in SEQ ID NO:
 30. 87. The method of claim 75,wherein the antibody comprises a VH region comprising any one of thesequences as set forth in SEQ ID NOs: 2, 7, 12, 17, and 22, and a VLregion comprising any one of the sequences as set forth in SEQ ID NOs:27, 32, 37, 42, and
 47. 88. The method of claim 75, wherein the antibodycomprises: (i) a VH region comprising the sequence as set forth in SEQID NO: 2, and a VL region comprising the sequence as set forth in SEQ IDNO: 27, (ii) a VH region comprising the sequence as set forth in SEQ IDNO: 7, and a VL region comprising the sequence as set forth in SEQ IDNO: 32, (iii) a VH region comprising the sequence as set forth in SEQ IDNO: 12, and a VL region comprising the sequence as set forth in SEQ IDNO: 37, (iv) a VH region comprising the sequence as set forth in SEQ IDNO: 17, and a VL region comprising the sequence as set forth in SEQ IDNO: 42, or (v) a VH region comprising the sequence as set forth in SEQID NO: 22, and a VL region comprising the sequence as set forth in SEQID NO:
 47. 89. The method of claim 75, wherein the antibody comprises aVH region comprising the sequence as set forth in SEQ ID NO: 2, and a VLregion comprising the sequence as set forth in SEQ ID NO:
 27. 90. Themethod of claim 75, wherein the antibody possesses the following bindingcharacteristics: (i) it does not bind to a variant of human CD38 whereinAsp in position 202 has been substituted with Gly to the same degreethat it binds to human CD38, (ii) it binds to a variant of human CD38wherein Gln in position 272 has been substituted with Arg to the samedegree that it binds to human CD38, and (iii) it binds to a variant ofhuman CD38 wherein the Ser in position 274 has been substituted with Pheto the same degree that it binds to human CD38.
 91. The method of claim75, wherein the antibody binds to human CD38 with a K_(D) of 10⁻⁸ M orless.
 92. The method of claim 75, wherein the antibody is a full lengthIgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody.
 93. The methodof claim 75, wherein the antibody is an antibody fragment or asingle-chain antibody.
 94. The method of claim 75, wherein the antibodyis an effector function-deficient antibody.
 95. The method of claim 75,wherein the antibody is a monovalent antibody.
 96. The method of claim75, wherein the antibody is a human monovalent antibody.
 97. The methodof claim 75, wherein the antibody is a bivalent antibody.